Methods and compositions to inhibit metastasis and to treat fibrosis and to enhance wound healing

ABSTRACT

Methods and compositions are provided for inhibiting or treating metastasis based on discoveries regarding Kif19 and Cep192. Methods and compositions are provided for enhancing wound healing, treating fibrosis, reducing scarring and treating nerve pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.15/706,849, filed Sep. 18, 2017, now allowed, which is a divisional ofU.S. application Ser. No. 15/023,869, filed Mar. 22, 2016, now U.S. Pat.No. 9,994,845, issued Jun. 12, 2018, U.S. national stage entry under 35U.S.C. § 371 of PCT International Patent Application No.PCT/US2014/055393, filed Sep. 12, 2014, which claims benefit of U.S.Provisional Application No. 61/885,676, filed Oct. 2, 2013, the contentsof each of which are incorporated herein by reference into the subjectapplication.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberW81XWH1210379 awarded by the Telemedicine and Advanced TechnologyResearch Center (TATRC) at the U.S. Army Medical Research and MaterielCommand (USAMRMC). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The disclosures of all publications, patents, patent applicationpublications and books referred to in this application are herebyincorporated by reference in their entirety into the subject applicationto more fully describe the art to which the subject invention pertains.

Cancer metastasis is stimulated by the movement of cancer cells from theprimary tumor to other tissues or organs. Metastatic cancer isresponsible for the majority of cancer deaths. There are currently noeffective means of treating metastasis, so the development of agentsthat inhibit the ability of cancer cells to move along their substratafor treating or inhibiting metastasis would represent a major advance.

Fibrosis is the formation of excess fibrous connective tissue in anorgan or tissue in a reparative or reactive process. This results fromthe hyperproliferation and motility of cells, such as fibroblasts, thatlay down connective tissue. Fibrosis can be a reactive, benign, orpathological state. In response to injury this is called scarring and iffibrosis arises from a single cell line this is called a fibroma.Physiologically this acts to deposit connective tissue, which canobliterate the architecture and function of the underlying organ ortissue. Fibrosis can be used to describe the pathological state ofexcess deposition of fibrous tissue, as well as the process ofconnective tissue deposition in healing. Fibrosis is similar tometastasis in that there are currently few therapeutic treatmentstrategies. The development of agents that prevent cell motility intowounded tissue would represent an important advance. Related to this,the development of safe and effective therapies for treating acute andchronic wounds is also of great interest. Wound healing is an intricate,multi-stage process that relies heavily on the delivery of new cells tothe wound zone. Two key elements of the wound healing response arefibroplasia and epithelialization when fibroblasts and epithelial cells,respectively, enter the wound to form a protective barrier from theexternal environment. This is stimulated by cell proliferation andmigration from the wound edge. The identification of agents thatincrease the rate at which cells invade and close a wound wouldrepresent a major advance in wound healing therapeutics. Ideally, thiswould be a topically applied agent that stimulates the proliferation andmigration of fibroblasts and wound edge epithelial cells.

The present invention addresses this need and identifies novel targetsin treating and preventing metastasis, treating and preventing fibrosis,and treating and preventing pain associated with wound healing.

SUMMARY OF THE INVENTION

A method of treating metastasis or inhibiting metastasis in a subjecthaving a cancer is provided comprising administering to the subject anamount of an inhibitor of KIF19 or of Kif19 gene product effective totreat metastasis or inhibit metastasis.

Also provided is a method of treating metastasis or inhibitingmetastasis in a subject having a cancer comprising administering to thesubject an amount of an inhibitor of CEP192 or of Cep192 gene producteffective to treat metastasis or inhibit metastasis.

Also provided is a method of treating fibrosis or scarring, or ofinhibiting fibrosis or scarring, in a subject in need thereof comprisingadministering to the subject an amount of an inhibitor of KIF19 or ofKif19 gene product effective to treat fibrosis or scarring, or inhibitfibrosis or scarring.

Also provided is a method of treating fibrosis or scarring, orinhibiting fibrosis or scarring, in a subject in need thereof comprisingadministering to the subject an amount of an inhibitor of Cep192effective to treat fibrosis or scarring, or inhibit fibrosis orscarring.

Also provided is a method of treating pain associated with wound healingin a subject having a wound comprising administering to the subject anamount of an inhibitor of Cep192 effective to treat pain associated withwound healing.

Also provided is an inhibitor of KIF19, or of Kif19 gene product isprovided for treating metastasis or inhibiting metastasis in a subjecthaving a cancer.

Also provided is an inhibitor of CEP192 or of Cep192 gene product isprovided for treating metastasis or inhibiting metastasis in a subjecthaving a cancer.

Also provided is an inhibitor of KIF19, or of Kif19 gene product, isprovided for treating fibrosis or scarring in a subject in need thereof.

Also provided is an inhibitor of CEP192 or of Cep192 gene product, isprovided for treating fibrosis or scarring in a subject in need thereof.

Also provided is an inhibitor of CEP192 or of Cep192 gene product, fortreating pain associated with wound healing in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A confocal micrograph showing a human U2OS cell double-labeledfor Kif19 and the FA protein, vinculin. The far right panel is a highermagnification of the region boxed in “merge”.

FIG. 2: The images show regions of U2OS cells (human Osteosarcoma)immunostained for the focal adhesion protein vinculin. The depletion ofKif19 by siRNA induces a substantial increase in the size and number offocal adhesions particularly in the cell interior.

FIG. 3A-3C: Panel A shows fluorescence recovery after photobleaching(FRAP) of GFP-vinculin labeled focal adhesions from control and Kif19siRNA-treated U2OS cells. Time is shown in minutes: seconds. Panel Bshows a representative fluorescence recovery plot from each condition.Panel C plots the density of focal adhesions in untreated cells (pre)and at various time points after nocodazole washout. Repolymerization ofMTs after nocodazole washout was previously shown to stimulate focaladhesion disassembly (Ezratty, Partridge et al. 2005). The depletion ofKif19 prevents the disassembly of focal adhesions after nocodazolewashout.

FIG. 4: Shows siRNA depletion of Kif19 decreases the motility of cancercells in vitro.

FIG. 5: Shows an anaplastic thyroid carcinoma mouse model (Arciuch etal., Oncotarget, December 2011). Dissociated tumor is removed from mouseand bathed in nanoparticles containing control or Kif19 siRNA for 2-24hrs. Tumors are then embedded in matrigel and imaged daily. Movement ofcells from tumor into matrigel is considered invasion. Black dots movingaway from the dark central mass are invasive tumor cells. Kif19nanoparticle siRNA treatment reduces tumor cell invasion relative tocontrols.

FIG. 6: Time-series of TIRF images showing a field offluorescently-labeled taxol-stabilized microtubules incubated withpurified recombinant full-length Kif19. The time from the first to lastimage is 5 minutes.

FIG. 7A-7F: Kif19 is a microtubule depolymerase that localizes tosubstrate adhesions and promotes cell motility. A) Time series images ofa fluorescent microtubule incubated with purified recombinant Kif19. B)Immunofluorescence showing the co-localization of Kif19 with the focaladhesion protein, vinculin. C) High magnification image showing a regionof a cell double-labeled for Kif19 and microtubules. Microtubules oftenterminate at kif19-labeled substrate adhesions and these interactionsare believed to control adhesion turnover rate. D) Control and Kif19siRNA treated human U2OS cells labeled for the focal adhesion marker,vinculin. In cells depleted of Kif19, adhesions become significantlyenlarged and hyperstable. E) Measured rates of in vitro wound closure incontrol and Kif19 siRNA-treated cultures (scratch assay). F) Movementtrajectories of control and Kif19 siRNA-treated cells plotted from acommon origin. The loss of Kif19 nearly completely suppresses cellmovement.

FIG. 8: Confocal micrographs showing control and Cep192 siRNA-treatedU2OS cells immunolabeled for microtubules (red) and Cep192 (green).Cep192 siRNA treatment eliminates Cep192 immunofluorescence indicating astrong protein knockdown. Controls showed robust centrosomes with radialMT arrays while Cep192-depleted cells contained non-radial MTarrangements. Inset shows higher magnification of boxed region.

FIG. 9A-B: U2OS cells were treated with siRNA for 72 hours then exposedto 5 uM nocodazole for 1 hour to depolymerize microtubules. Cells werethen washed 3× with warm DMEM and then incubated for 10 minutes to allowmicrotubule regrowth. Images show control and Cep192 siRNA treated cellsstained for microtubules. B) Control cells showed a significantly higheramount of regrowth from the centrosome than did cells depleted ofCep192; P<0.0001. S.E.M. is depicted as vertical bars.

FIG. 10A-10D: A) Time-lapse phase-contrast images of control and Cep192siRNA treated U2OS cells from an in vitro wound healing assay. U2OScells were plated into Ibidi Culture-Insert dishes following knockdown.B) Significantly fewer Cep192 depleted cells entered the wound zonerelative to controls. P<0.0001. S.E.M. is depicted as vertical bars. C)Time-lapse phase-contrast images of control and Cep192 siRNA-treatedHEKa (human epidermal keratinoctyes—adult) cells from an in vitro woundhealing assay. HEKa cells were plated into Ibidi Culture-Insert dishesfollowing. D) Significantly fewer Cep192-depleted cells entered thewound zone relative to controls. P<0.0001. S.E.M. is depicted asvertical bars.

FIG. 11: Anaplastic thyroid carcinoma invasion assay: a dissociatedtumor is removed from mouse and bathed in nanoparticles for 2 hrs. (48hours) (top Panels). The tumor is embedded in Matrigel/Collagen matrixand imaged daily (96 hours) (bottom panels).

FIG. 12: Human large cell lung tumors. SC injection of human H460 lungcancer cells into mice (top panels). Invasive tumors (1-2 cm) removedand bathed in nanoparticles for 2 hrs. (middle panels). Tumor embeddedin Matrigel/Collagen matrix and imaged daily (bottom panels).

FIG. 13: Fidgetin and Cep192 regulate axon regeneration. Images areimmunofluorescence micrographs of primary adult rat DRG neurons treatedwith control, Fidgetin or Cep192 nanoparticle encapsulated siRNA. Cellswere fixed 24 hours after plating and siRNA treatment. Bottom rightpanel shows the average axon length in each condition (longest processfrom each individual cell was measured; error bars are SEM). ***P<0.01;**P<0.05.

FIG. 14: Angiogenesis of fetal hearts 48 hours after treatment—Imagesshow representative control and Cep192 siRNA treated hearts two daysafter siRNA treatment. In the control, migrating endocaridal cells havepenetrated the ventricular wall and formed a fine vascular network. Bycontrast, Cep192 siRNA treated hearts have no apparent vessels at thissame time point. Thus, the depletion of Cep192 dramatically inhibits theangiogenic process by the endocardial cells.

DETAILED DESCRIPTION OF THE INVENTION

A method of treating metastasis or inhibiting metastasis in a subjecthaving a cancer is provided comprising administering to the subject anamount of an inhibitor of KIF19 or of Kif19 gene product effective totreat metastasis or inhibit metastasis.

As used herein, “treating” metastasis means ameliorating or lessening orreducing further progression of an extant metastasis. As used herein,“inhibiting” metastasis means lessening the extent of, development of,or progression of a new metastasis.

In embodiments of the invention described herein where both treating andinhibting a condition are recited, the individual embodiments oftreating and inhibiting are also encompassed separately. Thus, methodsof treating are provided. And methods of inhibiting are also separatelyprovided.

In an embodiment, the preferred subject is a human subject.

In embodiments of the invention described herein, the preferred subjectis a human subject.

Also provided is a method of treating metastasis or inhibitingmetastasis in a subject having a cancer comprising administering to thesubject an amount of an inhibitor of CEP192 or of Cep192 gene producteffective to treat metastasis or inhibit metastasis.

Also provided is a method of treating fibrosis or scarring, or ofinhibiting fibrosis or scarring, in a subject in need thereof comprisingadministering to the subject an amount of an inhibitor of KIF19 or ofKif19 gene product effective to treat fibrosis or scarring, or inhibitfibrosis or scarring. As used herein, “treating” a fibrosis meansameliorating or lessening or reducing further progression of an extantfibrosis. As used herein, “inhibiting” fibrosis means lessening theextent of, development of, or progression of a fibrosis. As used herein,“treating” scarring means ameliorating or lessening or reducing furtherprogression of an extant scarring or scarring process. As used herein,“inhibiting” scarring means lessening the extent of, development of, orprogression of a scarring or scarring process.

As used herein, any recitation of embodiments in the alternative, e.g.embodiment A or embodiment B, includes the specific, separateembodiments of (i) embodiment A and (ii) of embodiment B, as part of theinvention.

Also provided is a method of treating fibrosis or scarring, orinhibiting fibrosis or scarring, in a subject in need thereof comprisingadministering to the subject an amount of an inhibitor of CEP192 or ofan inhibitor of Cep192 gene product effective to treat fibrosis orscarring, or inhibit fibrosis or scarring.

Also provided is a method of treating pain associated with wound healingin a subject having a wound comprising administering to the subject anamount of an inhibitor of CEP192 or of an inhibitor of Cep192 geneproduct effective to treat pain associated with wound healing. As usedherein, “treating” pain associated with wound healing means amelioratingor lessening or reducing pain associated with an extant wound.

In an embodiment of the methods, the KIF19 or Kif19 gene product is ahuman KIF19 or human Kif19 gene product, respectively.

In an embodiment of the methods, the CEP192 or Cep192 gene product is ahuman CEP192 or a human Cep192 gene product, respectively.

In an embodiment of the methods, the inhibitor of KIF19 is an RNAinucleic acid. In an embodiment of the methods, the inhibitor of CEP192is an RNAi nucleic acid. In an embodiment of the methods, the RNAinucleic acid is a siRNA directed to KIF19 or a shRNA directed to KIF19.In an embodiment of the methods, the RNAi nucleic acid is a siRNAdirected to CEP192 or a shRNA directed to CEP192. In an embodiment ofthe methods, the siRNA is administered. In an embodiment of the methods,the shRNA is administered. In an embodiment of the methods, the siRNA isadministered as a composition comprising the siRNA associated with ananoparticle. In an embodiment of the methods, the siRNA is administeredas a composition comprising the siRNA encapsulated with a nanoparticle.In an embodiment of the methods, the nanoparticle is PEGylated. In anembodiment of the methods, the siRNA is administered as a viral vector.In an embodiment of the methods, the shRNA is administered as a viralvector.

In an embodiment of the methods, the cancer is a thyroid, blood,bladder, breast, colorectal, kidney, lung, melanoma, ovary, pancreas,prostate or stomach cancer. In an embodiment of the methods, the canceris an anaplastic thyroid carcinoma. In an embodiment of the methods, thecancer is large cell lung cancer.

In an embodiment of the methods, the fibrosis is in response to aninjury. In an embodiment of the methods, the fibrosis is a fibroma,pulmonary fibrosis, cystic fibrosis, hepatic cirrhosis, endomyocardialfibrosis, from a previous myocardial infarction, atrial fibrosis,mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis of the lungs, a complication ofpneumoconiosis, nephrogenic systemic fibrosis, Crohn's disease fibrosis,keloid fibrosis, scleroderma/systemic sclerosis of skin or lungs,arthrofibrosis or adhesive capsulitis fibrosis.

In an embodiment of the methods, the scarring is skin scarring,cardiovascular scarring, cardiac tissue scarring, or neuronal scarring.

In an embodiment of the methods, the wound is a skin wound,cardiovascular wound, a cardiac tissue wound, or neuronal wound. In anembodiment of the methods, the skin wound is a burn wound.

In an embodiment of the methods regarding wounds, scarring or treatingpain associated with the wound, the inhibitor may be applied directly tothe wound of the subject.

In an embodiment of the methods regarding skin wounds, scarring ortreating pain associated with the skin wound, the inhibitor may beapplied to the skin of the subject.

Also provided is a method of identifying an anti-metastatic agentcomprising contacting a nucleic acid encoding Kif19 gene product withthe agent or contacting Kif19 gene product with the agent anddetermining if the agent inhibits expression of the nucleic acid-encodedKif19 gene product or inhibits activity of the Kif19 gene product,respectively, and subsequently identifying the agent as ananti-metastatic agent or not, wherein an agent that inhibits Kif19expression or Kif19 gene product is identified as an anti-metastaticagent.

Preferably, an “agent” in the methods of identifying an anti-metastaticagent, anti-fibrotic agent, or pain-relieving agent, is a small organicmolecule of 1,500 daltons or less, a peptide, a protein, an antibody, afragment of an antibody, a carbohydrate, an oligonucleotide or a nucleicacid. In an embodiment of the methods of identifying an agent as setforth herein, the agent is a small organic molecule, a peptide, anucleic acid, an oligonucleotide, an antibody, an antigen-bindingfragment of an antibody or an aptamer.

Also provided is a method of identifying an anti-metastatic agentcomprising contacting a nucleic acid encoding Cep192 gene product withthe agent or contacting Cep192 gene product with the agent anddetermining if the agent inhibits expression of the nucleic acid-encodedCep192 gene product or inhibits activity of the Cep192 gene product,respectively, and subsequently identifying the agent as ananti-metastatic agent or not, wherein an agent that inhibits Cep192expression or Cep192 gene product is identified as an anti-metastaticagent.

Also provided is a method of identifying an anti-fibrotic agentcomprising contacting a nucleic acid encoding Kif19 gene product withthe agent or contacting Kif19 gene product with the agent anddetermining if the agent inhibits expression of the nucleic acid-encodedKif19 gene product or inhibits activity of the Kif19 gene product,respectively, and subsequently identifying the agent as an anti-fibroticagent or not, wherein an agent that inhibits Kif19 expression or Kif19gene product is identified as an anti-fibrotic agent.

Also provided is a method of identifying an anti-fibrotic agentcomprising contacting a nucleic acid encoding Cep192 gene product withthe agent or contacting Cep192 gene product with the agent anddetermining if the agent inhibits expression of the nucleic acid-encodedCep192 gene product or inhibits activity of the Cep192 gene product,respectively, and subsequently identifying the agent as an anti-fibroticagent or not, wherein an agent that inhibits Cep192 expression or Cep192gene product is identified as an anti-fibrotic agent.

Also provided is a method of identifying a pain-relieving agentcomprising contacting a nucleic acid encoding Cep192 gene product withthe agent or contacting Cep192 gene product with the agent anddetermining if the agent inhibits expression of the nucleic acid-encodedCep192 gene product or inhibits activity of the Cep192 gene product,respectively, and subsequently identifying the agent as a pain-relievingagent or not, wherein an agent that inhibits Cep192 expression or Cep192gene product is identified as a pain-relieving agent.

Generally herein, with regard to KIF19 and Kif19, “KIF19” (i.e. uppercase) refers to the gene and “Kif19” (i.e. lower case) refers to theprotein. The protein may also be referred to as “Kif19 gene product.”Generally herein, with regard to CEP192 and Cep192, “CEP192” (i.e. uppercase) refers to the gene and “Cep192” (i.e. lower case) refers to theprotein. The protein may also be referred to as “Cep192 gene product.”As used herein, a transcript of a given gene means any nucleic acid, forexample an mRNA, that encodes the protein gene product encoded by thegene. Thus, a transcript of CEP192 includes an mRNA encoding CEP192 geneproduct. Thus, a transcript of KIF19 includes an mRNA encoding KIF19gene product.

A pharmaceutical composition is provided comprising an amount of aninhibitor of KIF19 or of Kif19 gene product. In an embodiment, thepharmaceutical composition comprises an amount of an inhibitor of KIF19or of Kif19 gene product effective to treat a wound in a human subject,or comprises an amount of an inhibitor of KIF19 or of Kif19 gene producteffective to treat or inhibit metastasis in a subject, or comprises anamount of an inhibitor of KIF19 or of Kif19 gene product effective totreat or inhibit fibrosis in a subject. In an embodiment, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier. In an embodiment of the pharmaceutical composition, theinhibitor of KIF19 or of Kif19 gene product is encapsulated, completelyor partially, by a nanoparticle. In an embodiment the nanoparticlecomprises a hydrogel/sugar glass composite. In an embodiment, thenanoparticle is PEGylated. In an embodiment the nanoparticle is aliposomal nanoparticle. In an embodiment, the nanoparticle isparamagnetic. In an embodiment of the methods and compositions, theinhibitor is an siRNA which inhibits expression of Kif19 gene product.In an embodiment, the inhibitor is an shRNA which inhibits expression ofKif19 gene product.

The optimal dosage of the KIF19 inhibitor or of Kif19 gene productinhibitor administered in treatments herein will vary depending uponfactors such as the pharmacodynamic characteristics of a specificinhibitor and its mode and route of administration; the age, sex,metabolic rate, absorptive efficiency, health and weight of therecipient; the nature and extent of the symptoms; the kind of concurrenttreatment being administered; the frequency of treatment with theinhibitor and the desired therapeutic effect. A dosage unit of the KIF19inhibitor or of Kif19 gene product inhibitor may comprise a singlecompound, or a mixture of the compound with one or more anti-infectioncompound(s) or wound healing-promoting compound(s); one or moreanti-cancer compounds; or one or more anti-fibrotic compounds, asrelevant to the condition being treated.

In an embodiment of the methods or compositions, inhibition is effectedby RNAi. In an embodiment, RNAi inhibition of KIF19 or of Kif19 geneproduct expression is effected with an siRNA. The siRNA (smallinterfering RNA) with regard to KIF19/kif19 gene product as used in themethods or compositions described herein comprises a portion which iscomplementary to a nucleic acid sequence (in a non-limiting example anmRNA) encoding a Kif19 gene product. In an embodiment, the Kif19 geneproduct is a human Kif19 gene product. In an embodiment, the mRNA is oris encoded by NCBI Reference Sequence: NM_153209.3 (SEQ ID NO:1), andthe siRNA is effective to inhibit expression of Kif19 gene product. Inan embodiment, the mRNA is or is encoded by a known variant of the NCBIReference Sequence: NM_153209.3 (SEQ ID NO:1), and the siRNA iseffective to inhibit expression of Kif19 gene product. In an embodiment,the Kif19 gene product comprises consecutive amino acid residues havingthe sequence set forth in SEQ ID NO:2.

In an embodiment, the siRNA with regard to KIF19/kif19 gene productcomprises a double-stranded portion (duplex). Tn an embodiment, thesiRNA is 20-25 nucleotides in length. In an embodiment the siRNAcomprises a 19-21 core RNA duplex with a one or two nucleotide 3′overhang on, independently, either one or both strands. The siRNA can be5′ phosphorylated, or not, and may be modified with any of the knownmodifications in the art to improve efficacy and/or resistance tonuclease degradation. In an embodiment, the siRNA is 5′ phosphorylated.In an embodiment, the 5′ terminal residue of a strand of the siRNA isphosphorylated. In an embodiment the 5′ terminal residue of theantisense strand of the siRNA is phosphorylated. In one embodiment, asiRNA of the invention comprises a double-stranded RNA wherein onestrand of the double-stranded RNA is 80%, 85%, 90%, 95% or 100%complementary to a portion of an RNA transcript of a KIF19 (gene)encoding Kif19 gene product. In an embodiment, the RNA transcript of agene encoding Kif19 gene product is an mRNA. In an embodiment, the Kif19gene product is a human Kif19 gene product.

In an embodiment, a siRNA of the invention comprises a double-strandedRNA wherein one strand of the RNA comprises a portion having a sequencethe same as a portion of 18-25 consecutive nucleotides of an RNAtranscript of a gene encoding Kif19 gene product. In an embodiment, theother strand is fully complementary to the one strand. In an embodiment,the Kif19 gene product is a human Kif19 gene product. In yet anotherembodiment, a siRNA of the invention comprises a double-stranded RNAwherein both strands of RNA are connected by a non-nucleotide linker. Inyet another embodiment, a siRNA of the invention comprises adouble-stranded RNA wherein the two strands of RNA are not connectedother than by complementary hybridization. Alternately, a siRNA of theinvention comprises a double-stranded RNA wherein both strands of RNAare connected by a nucleotide linker, such as a loop or stem loopstructure. In an embodiment, one strand of the double-stranded siRNA isfully complementary to a nucleic acid encoding Kif19 gene product. In anembodiment, one strand of the double-stranded siRNA is fullycomplementary to a nucleic acid encoding Kif19 gene product except atone, or except at two, mismatched positions. In one embodiment, a singlestrand component of a siRNA of the invention is from 14 to 50nucleotides in length. In another embodiment, a single strand componentof a siRNA of the invention is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment,a single strand component of a siRNA of the invention is 18 nucleotidesin length. In yet another embodiment, a single strand component of asiRNA of the invention is 19 nucleotides in length. In yet anotherembodiment, a single strand component of a siRNA of the invention is 20nucleotides in length. In yet another embodiment, a single strandcomponent of a siRNA of the invention is 21 nucleotides in length. Inyet another embodiment, a single strand component of a siRNA of theinvention is 22 nucleotides in length. In yet another embodiment, asingle strand component of a siRNA of the invention is 23 nucleotides inlength. In one embodiment, a siRNA of the invention is from 28 to 56nucleotides in length. In another embodiment, a siRNA of the inventionis 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides inlength. In another embodiment, an siRNA of the invention comprises atleast one 2′-sugar modification. In an embodiment, an siRNA of theinvention comprises at least one nucleic acid base modification. In anembodiment, an siRNA of the invention comprises at least one phosphatebackbone modification. As used herein, “at least one” means one or more.In an embodiment, the double-stranded siRNA of the invention comprisesan overhang of one or two nucleotides. In an embodiment, the overhang isa 3′ overhang. In an embodiment, the overhang is a 5′ overhang. In anembodiment, the overhang is a 3′ overhang of two nucleotides. In anembodiment, the overhang is one of UU, UG or dTdT. In an embodiment, thedouble-stranded siRNA of the invention comprises an overhang of one ortwo nucleotides on each of its two strands. In an embodiment, the twooverhangs are 3′ overhangs. In an embodiment, the two overhangs are ofone nucleotide each. In an embodiment, the two overhangs are of twonucleotides each. In an embodiment, the overhangs are one of UU, UG ordTdT. In an embodiment, the 5′ terminal residue of a strand of the siRNAis phosphorylated. In an embodiment the 5′ terminal residue of theantisense strand of the siRNA is phosphorylated.

In one embodiment, RNAi inhibition of KIF19 or of Kif19 gene productexpression is effected by a short hairpin RNA (“shRNA”). The shRNA isintroduced into the appropriate cell by transduction with a vector. Inan embodiment, the vector is a lentiviral vector. In an embodiment, thevector comprises a promoter. In an embodiment, the promoter is a U6 orH1 promoter. In an embodiment the shRNA encoded by the vector is a firstnucleotide sequence ranging from 19-29 nucleotides complementary to thetarget gene/mRNA, in the present case the mRNA encodes Kif19 geneproduct. In an embodiment the Kif19 gene product is a human Kif19 geneproduct. In an embodiment the shRNA encoded by the vector also comprisesa short spacer of 4-15 nucleotides (a loop, which does not hybridize)and a 19-29 nucleotide sequence that is a reverse complement of thefirst nucleotide sequence. In an embodiment the siRNA resulting fromintracellular processing of the shRNA has overhangs of 1 or 2nucleotides. In an embodiment the siRNA resulting from intracellularprocessing of the shRNA overhangs has two 3′ overhangs. In an embodimentthe overhangs are, independently, UU, UG or dTdT.

In a preferred embodiment, the inhibitor of KIF19 or of Kif19 geneproduct expression is an siRNA. In a preferred embodiment the siRNA isencapsulated in a nanoparticle. In an embodiment, the nanoparticlecomprises a hydrogel/sugar glass composite. In an embodiment thenanoparticle is a liposomal nanoparticle. In an embodiment, thenanoparticle is PEGylated. In embodiments the PEG is PEG-500 or PEG-3000or PEG-5000. In an embodiment, the nanoparticle is doped with aminosilanes. In an embodiment, the nanoparticle is paramagnetic.

In embodiments, the siRNA, or the shRNA, (the KIF19 siRNA, the KIF19shRNA, the CEP192 siRNA or the CEP192 shRNA, or the gene product siRNAsor shRNAs) is modified at a 2 position of a sugar of at least onenucleotide thereof of at least one strand thereof. In an embodiment themodification is on a guide strand thereof. In an embodiment, the shRNAis modified. In an embodiment, the siRNA is modified. In an embodiment,the modification is a 2′-OMe modification. In an embodiment, themodification is a 2′-F modification. In an embodiment, the modificationis a 2′-O-benzyl modification. In an embodiment, the modification is a2′-O-methyl-4-pyridine (2′-O—CH₂Py(4)) modification.

As used herein an “aptamer”, with regard to KIF19 or Kif19, is asingle-stranded oligonucleotide or oligonucleotide analog that binds toa Kif19 gene product, or to a nucleic acid (such as KIF19) encoding aKif19 gene product, and inhibits the function or expression thereof, asappropriate.

The present invention provides kits for treating wounds or scarring, akit for treating or inhibiting metastasis, a kit for treating orinhibiting fibrosis, the kit comprising an inhibitor of KIF19 or aninhibitor of Kif19.

A composition provided in such a kit for treating or inhibitingmetastasis may be provided in a form suitable for reconstitution priorto use (such as a lyophilized injectable composition) or in a form whichis suitable for immediate application by, for example, injection, suchas an aqueous composition.

A composition provided in such a kit for treating wounds or scarring maybe provided in a form suitable for reconstitution prior to use (such asa lyophilized injectable composition) or in a form which is suitable forimmediate application to a wound, including to the wound margin, such asa lotion or ointment. In an embodiment for treating wounds, theinhibitor of KIF19 or of Kif19 gene product is administered locally tothe wound.

In an embodiment, the inhibitor of KIF19 or of Kif19 product isadministered via a vein or artery. In an embodiment, the inhibitor ofKIF19 or of Kif19 gene product is administered by injection,catheterization or cannulation.

In an embodiment, the inhibitor of KIF19 or of Kif19 gene product isadministered from an implant that elutes the inhibitor, for example aeluting stent or an eluting skin patch.

In an embodiment, the wound is an epidermal wound. In an embodiment, thewound is a skin wound. In an embodiment, the wound is a cardiac tissuewound. In an embodiment, the wound is a cardiovascular wound, forexample resulting from a myocardial infarction. In an embodiment, thewound is a neuronal wound. In an embodiment for treating wounds, theinhibitor of Kif19 is provided by a subcutaneous implant or depotmedicament system for the pulsatile delivery of the inhibitor to a woundor site where a wound is to expected be formed to promote wound healing.The inhibitor can be provided, for example, in a therapeuticallyeffective amount to each centimeter of a wound margin or each centimeterof a site at which a wound is expected to be formed. The benefits thatmay be derived from the present invention may be applicable to wounds atsites throughout the body. However, it may be preferred that the woundfor which healing is promoted is a skin wound. For illustrative purposesthe embodiments of the invention will generally be described withreference to skin wounds, although they remain applicable to othertissues and organs. Merely by way of example, in another preferredembodiment the wound may be a wound of the circulatory system,particularly of a blood vessel. Other wounds in which wound healing maybe promoted in accordance with the present invention include as a resultof surgery or as a result of a burn. Other wounds in which wound healingmay be promoted in accordance with the present invention include skinulcers caused by pressure, venous stasis, or diabetes mellitus. Examplesof specific wounds in which healing may be promoted using themedicaments and methods of treating wounds or promoting healing ofwounds described herein include, but are not limited to, thoseindependently selected from the group consisting of: wounds of the skin;wounds of the eye (including the inhibition of scarring resulting fromeye surgery such as LASIK surgery, LASEK surgery, PRK surgery, glaucomafiltration surgery, cataract surgery, or surgery in which the lenscapsule may be subject to scarring) such as those giving rise to cornealcicatrisation; wounds subject to capsular contraction (which is commonsurrounding breast implants); wounds of blood vessels; wounds of thecentral and peripheral nervous system (where prevention, reduction orinhibition of scarring may enhance neuronal reconnection and/or neuronalfunction); wounds of tendons, ligaments or muscle; wounds of the oralcavity, including the lips and palate (for example, to inhibit scarringresulting from treatment of cleft lip or palate); wounds of the internalorgans such as the liver, heart, brain, digestive tissues andreproductive tissues; wounds of body cavities such as the abdominalcavity, pelvic cavity and thoracic cavity (where inhibition of scarringmay reduce the number of incidences of adhesion formation and/or thesize of adhesions formed); and surgical wounds (in particular woundsassociated with cosmetic procedures, such as scar revision). It isparticularly preferred that the medicaments and methods of the inventionregarding wounds be used to promote healing of wounds of the skin.

A medicament in accordance with this aspect of the invention may beformulated in any appropriate carrier. Suitable carriers arepharmaceutically acceptable carriers, for example, preferably thoseconsistent with administration topically or administration by injectionfor treating wounds and treating or preventing fibrosis; preferablythose consistent with administration intravenously or administration byinjection or cannulation for treating or preventing metastasis. It willbe appreciated that, while the inhibitor of Kif19 may be administered bythe same route and in the same form in each incidence of treatment,different incidences of treatment may provide the inhibitor of Kif19 bydifferent medicaments and/or different routes of administration. Inembodiments of the invention the initial incidence of treatment mayprovide the inhibitor of Kif19 by means of an injection, such as anintradermal injection, while the second (and any subsequent) incidencesof treatment may involve provision of the inhibitor of Kif19 byalternative routes, such as topical formulations, or vice versa. In anembodiment, multiple administrations of the inhibitor of Kif19 may beeffected by the same means or route. In an embodiment the shRNA or siRNAinhibitor of Kif19 can be administered such that it is transfected intoone or more cells.

In a non-limiting embodiment the inhibitor of KIF 19 or Kif19 isprovided in a bulk-eroding system such as polylactic acid and glycolicacid (PLGA) copolymer based microspheres or microcapsules systemscontaining the inhibitor of Kif19. In an embodiment, blends ofPLGA:ethylcellulose systems may be used as an appropriate carrier. Afurther medicament in accordance with this aspect of the invention maybe formulated in a surface-eroding system wherein the inhibitor of Kif19or of KIF19 is embedded in an erodible matrix such as the poly(ortho)ester and polyanhydride matrices wherein the hydrolysis of the polymeris rapid. A medicament in accordance with this aspect of the inventionmay also be formulated by combining a pulsatile delivery system asdescribed above and an immediate release system such as a lyophilizedinjectable composition described above.

The inhibitor may be used in a composition with additives. Examples ofsuitable additives are sodium alginate, as a gelatinizing agent forpreparing a suitable base, or cellulose derivatives, such as guar orxanthan gum, inorganic gelatinizing agents, such as aluminum hydroxideor bentonites (termed thixotropic gel-formers), polyacrylic acidderivatives, such as Carbopol®, polyvinylpyrrolidone, microcrystallinecellulose and carboxymethylcellulose. Amphiphilic low molecular weightand higher molecular weight compounds, and also phospholipids, are alsosuitable. The gels can be present either as water-based hydrogels or ashydrophobic organogels, for example based on mixtures of low and highmolecular weight paraffin hydrocarbons and vaseline. The hydrophilicorganogels can be prepared, for example, on the basis of high molecularweight polyethylene glycols. These gelatinous forms are washable.Hydrophobic organogels are also suitable. Hydrophobic additives, such aspetroleum jelly, wax, oleyl alcohol, propylene glycol monostearateand/or propylene glycol monopalmitostearate, in particular isopropylmyristate can be included. In an embodiment the inhibitor is in acomposition comprising one or more dyes, for example yellow and/or rediron oxide and/or titanium dioxide for the purpose of matching asregards color. Compositions may be in any suitable form including gels,lotions, balms, pastes, sprays, powders, bandages, wound dressing,emulsions, creams and ointments of the mixed-phase or amphiphilicemulsion systems (oil/water-water/oil mixed phase), liposomes andtransfersomes or plasters/band aid-type coverings. Emulsifiers which canbe employed in compositions comprising the inhibitor of KIF19 or ofKif19 include anionic, cationic or neutral surfactants, for examplealkali metal soaps, metal soaps, amine soaps, sulphurated andsulphonated compounds, invert soaps, higher fatty alcohols, partialfatty acid esters of sorbitan and polyoxyethylene sorbitan, e.g. lanettetypes, wool wax, lanolin or other synthetic products for preparing theoil/water and/or water/oil emulsions.

Compositions comprising the inhibitor of Kif19 can also comprisevaseline, natural or synthetic waxes, fatty acids, fatty alcohols, fattyacid esters, for example as monoglycerides, diglycerides ortriglycerides, paraffin oil or vegetable oils, hydrogenated castor oilor coconut oil, hog fat, synthetic fats (for example based on caprylicacid, capric acid, lauric acid or stearic acid, such as Softisan®), ortriglyceride mixtures, such as Miglyol®, can be used as lipids, in theform of fatty and/or oleaginous and/or waxy components for preparing theointments, creams or emulsions of the compositions comprising theinhibitor of Kif19 used in the methods described herein.

Osmotically active acids and alkaline solutions, for examplehydrochloric acid, citric acid, sodium hydroxide solution, potassiumhydroxide solution, sodium hydrogen carbonate, may also be ingredientsof the compositions of the invention and, in addition, buffer systems,such as citrate, phosphate, tris buffer or triethanolamine, foradjusting the pH. It is possible to add preservatives as well, such asmethyl benzoate or propyl benzoate (parabens) or sorbic acid, forincreasing the stability.

Pastes, powders and solutions are additional forms of compositionscomprising the inhibitor of Kif19 which can be applied topically. Asconsistency-imparting bases, the pastes frequently contain hydrophobicand hydrophilic auxiliary substances, preferably, however, hydrophobicauxiliary substances containing a very high proportion of solids. Inorder to increase dispersity, and also flowability and slipperiness, andalso to prevent agglomerates, the powders or topically applicablepowders can, for example, contain starch species, such as wheat or ricestarch, flame-dispersed silicon dioxide or siliceous earth, which alsoserve as diluent.

A method is provided for identifying a candidate agent for treating awound comprising:

a) determining the activity of an amount of Kif19 gene product; and

b) contacting the amount of Kif19 gene product with the candidate agentand determining the activity of the amount of Kif19 gene product in thepresence of the candidate agent,

wherein a decreased activity of the amount of Kif19 gene product in thepresence of the candidate agent as compared to the activity of Kif19gene product in the absence of the candidate agent indicates that thecandidate agent can treat a wound, and wherein no change in or anincreased activity of the amount of Kif19 gene product in the presenceof the candidate agent as compared to the activity of Kif19 gene productin the absence of the candidate agent does not indicate that thecandidate agent can treat a wound. In an embodiment, the candidate agentis a small molecule of 2000 Daltons or less. In an embodiment, thecandidate agent is a small molecule of 1000 Daltons or less. In anembodiment, the candidate agent is a small molecule of 1500 Daltons orless. In an embodiment, the candidate agent is a substituted orun-substituted hydrocarbon small molecule. In an embodiment, theinhibitor or the candidate agent is an aptamer, a nucleic acid, anoligonucleotide, or a small organic molecule of 2000 Daltons or less. Inan embodiment, the inhibitor is cell-membrane permeable.

A pharmaceutical composition is provided comprising an amount of aninhibitor of CEP192 or of Cep192 gene product. In an embodiment, thepharmaceutical composition comprises an amount of an inhibitor of CEP192or of Cep192 gene product effective to treat a wound in a human subject,or comprises an amount of an inhibitor of CEP192 or of Cep192 geneproduct effective to treat or inhibit metastasis in a subject, orcomprises an amount of an inhibitor of CEP192 or of Cep192 gene producteffective to treat or inhibit fibrosis in a subject, or comprises anamount of an inhibitor of CEP192 or of Cep192 gene product effective totreat or inhibit pain associated with a wound or wound healing in asubject. In an embodiment, the pharmaceutical composition comprises apharmaceutically acceptable carrier. In an embodiment of thepharmaceutical composition, the inhibitor of CEP192 or of Cep192 geneproduct is encapsulated, completely or partially, by a nanoparticle. Inan embodiment the nanoparticle comprises a hydrogel/sugar glasscomposite. In an embodiment, the nanoparticle is PEGylated. In anembodiment the nanoparticle is a liposomal nanoparticle. In anembodiment, the nanoparticle is paramagnetic. In an embodiment of themethods and compositions, the inhibitor is an siRNA which inhibitsexpression of Cep192 gene product. In an embodiment, the inhibitor is anshRNA which inhibits expression of Cep192 gene product.

The optimal dosage of the CEP192 inhibitor or of Cep192 gene productinhibitor administered in treatments herein will vary depending uponfactors such as the pharmacodynamic characteristics of a specificinhibitor and its mode and route of administration; the age, sex,metabolic rate, absorptive efficiency, health and weight of therecipient; the nature and extent of the symptoms; the kind of concurrenttreatment being administered; the frequency of treatment with theinhibitor and the desired therapeutic effect. A dosage unit of theCEP192 inhibitor or of Cep192 gene product inhibitor may comprise asingle compound, or a mixture of the compound with one or moreanti-infection compound(s) or wound healing-promoting compound(s); oneor more anti-cancer compounds; or one or more anti-fibrotic compounds;or one or more pain-relieveing compounds, as relevant to the conditionbeing treated.

In an embodiment of the methods or compositions, inhibition of CEP192 orof Cep192 is effected by RNAi. In an embodiment, RNAi inhibition ofCEP192 or of Cep192 gene product expression is effected with an siRNA.The siRNA (small interfering RNA) as used in the methods or compositionsdescribed herein comprises a portion which is complementary to a nucleicacid, in a non-limiting example an mRNA, sequence encoding a Cep192 geneproduct. In an embodiment, the Cep192 gene product is a human Cep192gene product. Tn an embodiment, the mRNA is or is encoded by NCBTReference Sequence: NM_032142.3 (SEQ ID NO:3), and the siRNA iseffective to inhibit expression of Cep192 gene product. In anembodiment, the mRNA is or is encoded by a known variant of NCBIReference Sequence: NM_032142.3 (SEQ ID NO:3), and the siRNA iseffective to inhibit expression of Cep192 gene product. In anembodiment, the Cep192 gene product comprises consecutive amino acidresidues having the sequence set forth in SEQ ID NO:4.

In an embodiment, the siRNA with regard to CEP192/Cep192 gene productcomprises a double-stranded portion (duplex). In an embodiment, thesiRNA is 20-25 nucleotides in length. In an embodiment the siRNAcomprises a 19-21 core RNA duplex with a one or two nucleotide 3′overhang on, independently, either one or both strands. The siRNA can be5′ phosphorylated, or not, and may be modified with any of the knownmodifications in the art to improve efficacy and/or resistance tonuclease degradation. In an embodiment, the siRNA is 5′ phosphorylated.In an embodiment, the 5′ terminal residue of a strand of the siRNA isphosphorylated. In an embodiment the 5′ terminal residue of theantisense strand of the siRNA is phosphorylated. In one embodiment, asiRNA of the invention comprises a double-stranded RNA wherein onestrand of the double-stranded RNA is 80%, 85%, 90%, 95% or 100%complementary to a portion of an RNA transcript of a CEP192 (gene)encoding Cep192 gene product. In an embodiment, the RNA transcript of agene encoding Cep192 gene product is an mRNA. In an embodiment, theCep192 gene product is a human Cep192 gene product.

In an embodiment, a siRNA of the invention with regard to CEP192/Cep192gene product comprises a double-stranded RNA wherein one strand of theRNA comprises a portion having a sequence the same as a portion of 18-25consecutive nucleotides of an RNA transcript of a gene encoding Cep192gene product. In an embodiment, the other strand is fully complementaryto the one strand. In an embodiment, the Cep192 gene product is a humanCep192 gene product. In yet another embodiment, a siRNA of the inventioncomprises a double-stranded RNA wherein both strands of RNA areconnected by a non-nucleotide linker. In yet another embodiment, a siRNAof the invention comprises a double-stranded RNA wherein the two strandsof RNA are not connected other than by complementary hybridization.Alternately, a siRNA of the invention comprises a double-stranded RNAwherein both strands of RNA are connected by a nucleotide linker, suchas a loop or stem loop structure. In an embodiment, one strand of thedouble-stranded siRNA is fully complementary to a nucleic acid encodingCep192 gene product. In an embodiment, one strand of the double-strandedsiRNA is fully complementary to a nucleic acid encoding Cep192 geneproduct except at one, or except at two, mismatched positions. In oneembodiment, a single strand component of a siRNA of the invention isfrom 14 to 50 nucleotides in length. In another embodiment, a singlestrand component of a siRNA of the invention is 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In anembodiment, a single strand component of a siRNA of the invention is 18nucleotides in length. In yet another embodiment, a single strandcomponent of a siRNA of the invention is 19 nucleotides in length. Inyet another embodiment, a single strand component of a siRNA of theinvention is 20 nucleotides in length. In yet another embodiment, asingle strand component of a siRNA of the invention is 21 nucleotides inlength. In yet another embodiment, a single strand component of a siRNAof the invention is 22 nucleotides in length. In yet another embodiment,a single strand component of a siRNA of the invention is 23 nucleotidesin length. In one embodiment, a siRNA of the invention is from 28 to 56nucleotides in length. Tn another embodiment, a siRNA of the inventionis 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides inlength. In an embodiment, an siRNA of the invention comprises at leastone 2′-sugar modification. In an embodiment, an siRNA of the inventioncomprises at least one nucleic acid base modification. In an embodiment,an siRNA of the invention comprises at least one phosphate backbonemodification. As used herein, “at least one” means one or more. In anembodiment, the double-stranded siRNA of the invention comprises anoverhang of one or two nucleotides. In an embodiment, the overhang is a3′ overhang. In an embodiment, the overhang is a 5′ overhang. In anembodiment, the overhang is a 3′ overhang of two nucleotides. In anembodiment, the overhang is one of UU, UG or dTdT. In an embodiment, thedouble-stranded siRNA of the invention comprises an overhang of one ortwo nucleotides on each of its two strands. In an embodiment, the twooverhangs are 3′ overhangs. In an embodiment, the two overhangs are ofone nucleotide each. In an embodiment, the two overhangs are of twonucleotides each. In an embodiment, the overhangs are, independently,one of UU, UG or dTdT. In an embodiment, the 5′ terminal residue of astrand of the siRNA is phosphorylated. In an embodiment the 5′ terminalresidue of the antisense strand of the siRNA is phosphorylated.

In one embodiment, RNAi inhibition of CEP192 or of Cep192 gene productexpression is effected by a short hairpin RNA (“shRNA”). The shRNA isintroduced into the appropriate cell by transduction with a vector. Tnan embodiment, the vector is a lentiviral vector. In an embodiment, thevector comprises a promoter. In an embodiment, the promoter is a U6 orH1 promoter. In an embodiment the shRNA encoded by the vector is a firstnucleotide sequence ranging from 19-29 nucleotides complementary to thetarget gene/mRNA, in the present case the mRNA encodes Cep192 geneproduct. In an embodiment the Cep192 gene product is a human Cep192 geneproduct. In an embodiment the shRNA encoded by the vector also comprisesa short spacer of 4-15 nucleotides (a loop, which does not hybridize)and a 19-29 nucleotide sequence that is a reverse complement of thefirst nucleotide sequence. In an embodiment the siRNA resulting fromintracellular processing of the shRNA has overhangs of 1 or 2nucleotides. In an embodiment the siRNA resulting from intracellularprocessing of the shRNA overhangs has two 3′ overhangs. In an embodimentthe overhangs are, independently, UU, UG or dTdT.

As used herein an “aptamer”, with regard to CEP192 or Cep192, is asingle-stranded oligonucleotide or oligonucleotide analog that binds toa Cep192 gene product, or to a nucleic acid (such as CEP192) encoding aCep192 gene product, and inhibits the function or expression thereof, asappropriate.

The present invention provides kits for treating wounds or scarring, akit for treating or inhibiting metastasis, a kit for treating orinhibiting fibrosis, or a kit for treating or inhibiting pain associatedwith a wound or with wound healing, the kit comprising an inhibitor ofCEP192 or an inhibitor of Cep192.

A composition provided in such a kit for treating or inhibitingmetastasis may be provided in a form suitable for reconstitution priorto use (such as a lyophilized injectable composition) or in a form whichis suitable for immediate application by, for example, injection, suchas an aqueous composition.

A composition provided in such a kit for treating or inhibiting painassociated with a wound or with wound healing, may be provided in a formsuitable for reconstitution prior to use (such as a lyophilizedinjectable composition) or in a form which is suitable for immediateapplication by, for example, injection, such as an aqueous composition,or a form for immediate topical application, such as a lotion orointment.

A composition provided in such a kit for treating wounds or scarring maybe provided in a form suitable for reconstitution prior to use (such asa lyophilized injectable composition) or in a form which is suitable forimmediate application to a wound, including to the wound margin, such asa lotion or ointment. In an embodiment for treating wounds, theinhibitor of CEP192 or of Cep192 gene product is administered locally tothe wound.

In an embodiment, the inhibitor of CEP192 or of Cep192 product isadministered via a vein or artery. In an embodiment, the inhibitor ofCEP192 or of Cep192 gene product is administered by injection,catheterization or cannulation.

In an embodiment, the inhibitor of CEP192 or of Cep192 gene product isadministered from an implant that elutes the inhibitor, for example aeluting stent or an eluting skin patch.

In an embodiment, the wound is an epidermal wound. In an embodiment, thewound is a skin wound. In an embodiment, the wound is a cardiac tissuewound. In an embodiment, the wound is a cardiovascular wound, forexample resulting from a myocardial infarction. In an embodiment, thewound is a neuronal wound. In an embodiment of the invention theinhibitor of Cep192 is provided by a subcutaneous implant or depotmedicament system for the pulsatile delivery of the inhibitor to a woundor site where a wound is to expected be formed to promote wound healing.The inhibitor can be provided, for example, in a therapeuticallyeffective amount to each centimeter of a wound margin or each centimeterof a site at which a wound is expected to be formed. The benefits thatmay be derived from the present invention may be applicable to wounds atsites throughout the body. However, it may be preferred that the woundfor which healing is promoted is a skin wound. For illustrative purposesthe embodiments of the invention will generally be described withreference to skin wounds, although they remain applicable to othertissues and organs. Merely by way of example, in another preferredembodiment the wound may be a wound of the circulatory system,particularly of a blood vessel. Other wounds in which wound healing maybe promoted in accordance with the present invention include as a resultof surgery or as a result of a burn. Other wounds in which wound healingmay be promoted in accordance with the present invention include skinulcers caused by pressure, venous stasis, or diabetes mellitus. In anembodiment, the inhibitor of CEP192 or of Cep192 gene product isadministered locally to the wound. In an embodiment, the inhibitor ofCEP192 or of Cep192 gene product is administered via a vein or artery.In an embodiment, the inhibitor of CEP19 or of Cep19 gene product isadministered by injection, catheterization or cannulation. In anembodiment, the inhibitor of CEP192 or of Cep19 gene product isadministered from an implant that elutes the inhibitor, for example aeluting stent or an eluting skin patch. In an embodiment, the wound isan epidermal wound. In an embodiment, the wound is a skin wound. In anembodiment, the wound is a cardiac tissue wound. In an embodiment, thewound is a cardiovascular wound, for example resulting from a myocardialinfarction. In an embodiment, the wound is a neuronal wound. Examples ofspecific wounds in which healing may be promoted using the medicamentsand methods of treating wounds or promoting healing of wounds describedherein include, but are not limited to, those independently selectedfrom the group consisting of: wounds of the skin; wounds of the eye(including the inhibition of scarring resulting from eye surgery such asLASIK surgery, LASEK surgery, PRK surgery, glaucoma filtration surgery,cataract surgery, or surgery in which the lens capsule may be subject toscarring) such as those giving rise to corneal cicatrisation; woundssubject to capsular contraction (which is common surrounding breastimplants); wounds of blood vessels; wounds of the central and peripheralnervous system (where prevention, reduction or inhibition of scarringmay enhance neuronal reconnection and/or neuronal function); wounds oftendons, ligaments or muscle; wounds of the oral cavity, including thelips and palate (for example, to inhibit scarring resulting fromtreatment of cleft lip or palate); wounds of the internal organs such asthe liver, heart, brain, digestive tissues and reproductive tissues;wounds of body cavities such as the abdominal cavity, pelvic cavity andthoracic cavity (where inhibition of scarring may reduce the number ofincidences of adhesion formation and/or the size of adhesions formed);and surgical wounds (in particular wounds associated with cosmeticprocedures, such as scar revision). It is particularly preferred thatthe medicaments and methods of the invention regarding wounds be used topromote healing of wounds of the skin.

A medicament in accordance with this aspect of the invention may beformulated in any appropriate carrier. Suitable carriers arepharmaceutically acceptable carriers, for example, preferably thoseconsistent with administration topically or administration by injectionfor treating wounds and treating or preventing fibrosis; preferablythose consistent with administration intravenously or administration byinjection or cannulation for treating or preventing metastasis. It willbe appreciated that, while the inhibitor of Cep192 or CEP192 may beadministered by the same route and in the same form in each incidence oftreatment, different incidences of treatment may provide the inhibitorof Cep192 by different medicaments and/or different routes ofadministration. In embodiments of the invention the initial incidence oftreatment may provide the inhibitor of Cep192 by means of an injection,such as an intradermal injection, while the second (and any subsequent)incidences of treatment may involve provision of the inhibitor of Cep192by alternative routes, such as topical formulations, or vice versa. Inan embodiment, multiple administrations of the inhibitor of Cep192 maybe effected by the same means or route.

In an embodiment the shRNA or siRNA inhibitor of CEP192 or of Cep192gene product expression can be administered such that it is transfectedinto one or more cells.

In a preferred embodiment, the inhibitor is an siRNA. In a preferredembodiment the siRNA is encapsulated in a nanoparticle. In anembodiment, the nanoparticle comprises a hydrogel/sugar glass composite.In an embodiment the nanoparticle is a liposomal nanoparticle. In anembodiment, the nanoparticle is PEGylated. In embodiments the PEG isPEG-500 or PEG-3000 or PEG-5000. In an embodiment, the nanoparticle isdoped with amino silanes. In an embodiment, the nanoparticle isparamagnetic.

In a non-limiting embodiment the inhibitor of CEP192 or of Cep192 geneproduct is provided in a bulk-eroding system such as polylactic acid andglycolic acid (PLGA) copolymer based microspheres or microcapsulessystems containing the inhibitor of Cep192. In an embodiment, blends ofPLGA:ethylcellulose systems may be used as an appropriate carrier. Afurther medicament in accordance with this aspect of the invention maybe formulated in a surface-eroding system wherein the inhibitor ofCep192 is embedded in an erodible matrix such as the poly(ortho) esterand polyanhydride matrices wherein the hydrolysis of the polymer israpid. A medicament in accordance with this aspect of the invention mayalso be formulated by combining a pulsatile delivery system as describedabove and an immediate release system such as a lyophilized injectablecomposition described above.

The inhibitor may be used in a composition with additives. Examples ofsuitable additives are sodium alginate, as a gelatinizing agent forpreparing a suitable base, or cellulose derivatives, such as guar orxanthan gum, inorganic gelatinizing agents, such as aluminum hydroxideor bentonites (termed thixotropic gel-formers), polyacrylic acidderivatives, such as Carbopol®, polyvinylpyrrolidone, microcrystallinecellulose and carboxymethylcellulose. Amphiphilic low molecular weightand higher molecular weight compounds, and also phospholipids, are alsosuitable. The gels can be present either as water-based hydrogels or ashydrophobic organogels, for example based on mixtures of low and highmolecular weight paraffin hydrocarbons and vaseline. The hydrophilicorganogels can be prepared, for example, on the basis of high molecularweight polyethylene glycols. These gelatinous forms are washable.Hydrophobic organogels are also suitable. Hydrophobic additives, such aspetroleum jelly, wax, oleyl alcohol, propylene glycol monostearateand/or propylene glycol monopalmitostearate, in particular isopropylmyristate can be included. In an embodiment the inhibitor is in acomposition comprising one or more dyes, for example yellow and/or rediron oxide and/or titanium dioxide for the purpose of matching asregards color. Compositions may be in any suitable form including gels,lotions, balms, pastes, sprays, powders, bandages, wound dressing,emulsions, creams and ointments of the mixed-phase or amphiphilicemulsion systems (oil/water-water/oil mixed phase), liposomes andtransfersomes or plasters/band aid-type coverings. Emulsifiers which canbe employed in compositions comprising the inhibitor of CEP192 or ofCep192 include anionic, cationic or neutral surfactants, for examplealkali metal soaps, metal soaps, amine soaps, sulphurated andsulphonated compounds, invert soaps, higher fatty alcohols, partialfatty acid esters of sorbitan and polyoxyethylene sorbitan, e.g. lanettetypes, wool wax, lanolin or other synthetic products for preparing theoil/water and/or water/oil emulsions.

Compositions comprising the inhibitor ofCEP192 or of Cep192 can alsocomprise vaseline, natural or synthetic waxes, fatty acids, fattyalcohols, fatty acid esters, for example as monoglycerides, diglyceridesor triglycerides, paraffin oil or vegetable oils, hydrogenated castoroil or coconut oil, hog fat, synthetic fats (for example based oncaprylic acid, capric acid, lauric acid or stearic acid, such asSoftisant), or triglyceride mixtures, such as Miglyolg, can be used aslipids, in the form of fatty and/or oleaginous and/or waxy componentsfor preparing the ointments, creams or emulsions of the compositionscomprising the inhibitor of CEP192 or of Cep192 used in the methodsdescribed herein.

Osmotically active acids and alkaline solutions, for examplehydrochloric acid, citric acid, sodium hydroxide solution, potassiumhydroxide solution, sodium hydrogen carbonate, may also be ingredientsof the compositions of the invention and, in addition, buffer systems,such as citrate, phosphate, tris buffer or triethanolamine, foradjusting the pH. It is possible to add preservatives as well, such asmethyl benzoate or propyl benzoate (parabens) or sorbic acid, forincreasing the stability.

Pastes, powders and solutions are additional forms of compositionscomprising the inhibitor of Cep192 which can be applied topically. Asconsistency-imparting bases, the pastes frequently contain hydrophobicand hydrophilic auxiliary substances, preferably, however, hydrophobicauxiliary substances containing a very high proportion of solids. Inorder to increase dispersity, and also flowability and slipperiness, andalso to prevent agglomerates, the powders or topically applicablepowders can, for example, contain starch species, such as wheat or ricestarch, flame-dispersed silicon dioxide or siliceous earth, which alsoserve as diluent.

In an embodiment, insofar as the methods herein pertain to wounds orscarring, the compositions comprise further active ingredients suitablefor protecting or aiding in healing of the wound, for example one ormore antibiotics, antiseptics, vitamins, anesthetics, antihistamines,anti-inflammatory agents, moisturizers, penetration-enhancing agentsand/or anti-irritants.

In an embodiment of the methods and compositions described herein thesubject is a mammal. In an embodiment the subject is human.

As used herein, “promotion” of wound healing, or grammatical equivalent,means an acceleration in any one or more of visual appearance of woundrecovery, reduction in wound size, reduction in distance between woundmargins, scab formation, fibroplasia and re-epithelialization ascompared to the corresponding parameter in an untreated wound.

As used herein, “wound” is a break or discontinuity in the structure ofan organ or tissue (including skin), which includes epithelium,connective tissue, and muscle tissue, caused by an external agent.Examples of wounds include, but are not limited to, skin wounds,ulcerations, bedsores, grazes, tears, cuts, punctures, tympanic membraneperforations, burns, and those that are a consequence of plastic surgeryprocedures.

A method is provided for identifying a candidate agent for treating awound comprising:

a) determining the activity of an amount of Cep192 gene product; and

b) contacting the amount of Cep192 gene product with the candidate agentand determining the activity of the amount of Cep192 gene product in thepresence of the candidate agent,

wherein a decreased activity of the amount of Cep192 gene product in thepresence of the candidate agent as compared to the activity of Cep192gene product in the absence of the candidate agent indicates that thecandidate agent can treat a wound, and wherein no change in or anincreased activity of the amount of Cep192 gene product in the presenceof the candidate agent as compared to the activity of Cep192 geneproduct in the absence of the candidate agent does not indicate that thecandidate agent can treat a wound. In an embodiment, the candidate agentis a small molecule of 2000 Daltons or less. In an embodiment, thecandidate agent is a small molecule of 1000 Daltons or less. In anembodiment, the candidate agent is a small molecule of 1500 Daltons orless. In an embodiment, the candidate agent is a substituted orun-substituted hydrocarbon small molecule. In an embodiment, theinhibitor or the candidate agent is an aptamer, a nucleic acid, anoligonucleotide, or a small organic molecule of 2000 Daltons or less. Inan embodiment, the inhibitor is cell-membrane permeable.

With regard to the methods described herein to identify candidate agentsas inhibitors of Kif19 or of KTF19 of CEP192 or of Cep192, one skilledin the art can readily screen libraries of compounds, for example smallmolecule libraries, using the methods as described to identify agentswhich are inhibitors of Kif19 or of KIF19 of CEP192 or of Cep192 andwhich are therapeutic in treating wounds and promoting the healing ofwounds. In addition, one skilled in the art can employ the method toidentify peptides, peptidomimetics, antibodies, antibody fragments andnucleic acids which are inhibitors of Kif19 or of KIF19 of CEP192 or ofCep192 and which are therapeutic in treating wounds and promoting thehealing of wounds.

An inhibitor of KIF19, or of Kif19 gene product is provided for treatingmetastasis or inhibiting metastasis in a subject having a cancer.

An inhibitor of CEP192 or of Cep192 gene product is provided fortreating metastasis or inhibiting metastasis in a subject having acancer.

An inhibitor of KIF19, or of Kif19 gene product, is provided fortreating fibrosis or scarring in a subject in need thereof.

An inhibitor of CEP192 or of Cep192 gene product, is provided fortreating fibrosis or scarring in a subject in need thereof.

An inhibitor of CEP192 or of Cep192 gene product, for treating painassociated with wound healing in a subject.

In an embodiment, the inhibitor is an RNAi nucleic acid. In anembodiment, the inhibitor comprises an siRNA. In an embodiment, theinhibitor comprises an shRNA. In an embodiment, the siRNA or shRNA isdirected against CEP192. In an embodiment, the siRNA or shRNA isdirected against KIF19.

In an embodiment of the methods, prodcuts and compositions, theinhibitor is biomembrane-permeable or is conjugated or otherwiseattached to a moiety which renders the inhibitor biomembrane-permeable.

In an embodiment, KIF19 comprises the following sequence (SEQ ID NO:1):

1 gcgttgttgg tttcgggttg tcaggcagcg cgcgaggcgg cgggcagcta gcagctggcg 61gacgcgaccc ggaggcggtg ggggtgcggc tgagccatgc ccggtggcgc ggcctgagcc 121cctccacctg ctgcaatcat gaaggacagc ggggactcca aggaccagca actcatggtg 181gcgcttcggg tccggcccat cagcgtggca gagctggagg aaggagctac cctcatcgcc 241cataaagtgg atgagcagat ggtggttctc atggacccaa tggaggatcc cgacgacatc 301ctgcgggcgc atcgctcccg ggagaagtcc tacctgttcg acgtggcctt tgacttcacc 361gccacccagg agatggtgta tcaggccacc accaagagcc tcatcgaggg cgtcatctca 421ggctacaatg ccactgtctt tgcctatggc cccacaggct gtgggaaaac ctacaccatg 481ctgggcacag accaggagcc tggcatctat gttcagaccc tcaacgacct cttccgtgcc 541atcgaggaga ccagcaatga catggagtat gaggtctcca tgtcctacct ggagatctac 601aatgagatga tccgggacct gctgaacccc tccctgggct acctggagct gcgggaggac 661tctaaggggg tgatccaggt ggccggcatc accgaagtct ccaccatcaa tgccaaggag 721atcatgcagc tgctgatgaa ggggaaccgg cagaggaccc aggagcccac ggccgccaac 781cagacgtcct cccgctccca cgcggtactg caggtgaccg tgcgccagcg cagccgggtc 841aagaacatct tgcaggaggt gcggcagggc cgcctgttca tgatcgacct ggctggctca 901gagcgcgcct cgcagacaca gaatcgtggg cagcgtatga aggagggggc ccacatcaac 961cgctcactgc tggcactggg caactgcatc aacgccctga gcgacaaggg tagcaacaag 1021tacatcaact atcgcgacag caagctcacc cggctcctga aggactctct gggaggaaac 1081agccgcacag tgatgatcgc tcacatcagt cctgcgagca gtgccttcga ggagtcccgg 1141aacaccctga cctacgccgg ccgggccaag aacattaaga ctagggtgaa gcagaacctc 1201ctgaacgtct cctaccacat cgcccagtac accagcatca tcgctgacct gcggggcgag 1261atccagcgac tcaagcgcaa gattgatgag cagactgggc ggggccaggc ccggggccgg 1321caggatcggg gtgacatccg ccacatccaa gctgaggtcc agctgcacag cgggcagggt 1381gagaaggctg gcatgggaca gcttcgggag cagctcgcca gcgccttcca ggagcagatg 1441gatgtgcgga ggcgcctgct ggagctggag aaccgcgcca tggaggtcca gattgacacc 1501tcccgacacc tgctcaccat cgccggctgg aagcatgaga agtcccgccg ggccctcaaa 1561tggcgggagg agcagcgaaa ggagtgctac gctaaggacg acagcgagaa ggactcagac 1621acaggtgatg accaaccaga catcctggag ccacccgagg tggccgcagc ccgggagagc 1681attgcagccc tggtggacga gcagaagcaa ctgcgcaagc agaagctggc gctggagcag 1741cgctgccggg agctgcgcgc gcggggccgg cgcctggagg agacgctgcc gcggcgcatc 1801ggctccgagg agcagcgcga ggtgctcagc ctgctgtgcc gcgtgcacga gctcgaggtg 1861gagaacaccg agatgcagtc gcacgcgctg ctccgcgacg gtgcgctccg ccaccgccac 1921gaggccgtgc gccgcctgga gcagcaccgc agtctctgcg acgagattat ccagggccag 1981cggcagatca tcgacgacta caacctggcc gtcccgcagc gcctggaaga gctctacgaa 2041gtgtacctgc gggagctgga ggagggcagc ctggagcagg ccaccatcat ggaccaagtg 2101gcctccaggg ccctgcagga cagctccttg cccaaaatta ccccagcagg aacctcactg 2161accccagatt ctgacctgga gagtgtgaag acattgagct ctgatgccca gcacctgcag 2221aacagcgccc tccctcccct cagcacagag agtgaaggcc accacgtgtt caaggctggt 2281actggggcct ggcaggcaaa aagctectct gtgcccaccc cacctcccat ccagctcggc 2341agcctggtga cgcaggaggc cccggctcag gacagcctgg gcagctggat caactcttcc 2401cctgacagca gtgagaacct gtcggagatc cccttgtccc acaaagagag gaaggagatc 2461ctgactggca ccaagtgcat ctgggtgaag gccgcccggc ggcgctcgcg ggccctggga 2521accgaggggc gacacctgct ggcacccgcg acagagcgca gcagcctgtc cctgcactca 2581ctgagcgagg gcgacgatgc gcggccacca ggcccactgg cctgcaagcg gccgcccagc 2641cccacactac agcatgctgc cagtgaggac aacctgtcca gcagcacggg cgaggccccg 2701tcccgggcag tcggacatca tggggacggc cccaggccct ggctgcgtgg ccagaagaaa 2761agcctgggca agaaaaggga ggagtcgctg gaggcaaaga gaaggaagcg gaggtcccga 2821tccttcgagg tcaccgggca agggctctcc caccccaaga cacacctcct ggggccccat 2881caggcggagc gcatctcgga ccacaggatg ccagtgtgca ggcacccagc ccctggtatc 2941cggcatctgg gaaaggtcac gctacctttg gccaaagtca aactccctcc aagccagaac 3001acgggcccgg gggactcctc acccctggct gttcccccca acccaggtgg tggttctcga 3061cgggctaccc gtgggccccg cctgccccac ggcacaagca cccatggcaa agatggatgc 3121tcccggcata actgaggggc cctgcctgga actggctctc tcacctccca agactgaatg 3181gggtctagca gggcatggga ggtggaggct gggcagatgg agatgaccag gaagtaagct 3241caggatctca gcaggccagg gctcctgaga cccaggaact ggggtctctg cccaaccctc 3301ccatgctttc agtgccactg gggaaaagag gtgaggccag gggacatggc caggacggct 3361gggctccctg gcttcccagc cctggacaga atgctgttgc caaaacctgc acagccctga 3421ggccagcctc ggccttggta acggaggaaa gcagctgaca gtgagacggg gctcctggcc 3481cacgtgtggg gcacgggcat cctggatggt tggggaggcg ccgacaggca cttcacgtat 3541tacaattggg gatgtgggtg agggagggaa tctggItttg ttacttggca gtggtttttt 3601ctcacccttc ctttttaaca ataaaatccc atttgggtct tgaaaaaaaa aaaaaaaaaa 3661aaaaaaaaaa

In an embodiment, Kif19 gene product comprises the following sequence(SEQ ID NO:2):

MKDSGDSKDQQLMVALRVRPISVAELEEGATLIAHKVDEQMVVLMDPMEDPDDILRAHRSREKSYLFDVAFDFTATQEMVYQATTKSLIEGVISGYNATVFAYGPTGCGKTYTMLGTDQEPGIYVQTLNDLFRAIEETSNDMEYEVSMSYLEIYNEMIRDLLNPSLGYLELREDSKGVIQVAGITEVSTINAKEIMQLLMKGNRQRTQEPTAANQTSSRSHAVLQVTVRQRSRVKNILQEVRQGRLFMIDLAGSERASQTQNRGQRMKEGAHINRSLLALGNCINALSDKGSNKYINYRDSKLTRLLKDSLGGNSRTVMIAHISPASSAFEESRNTLTYAGRAKNIKTRVKQNLLNVSYHIAQYTSIIADLRGEIQRLKRKIDEQTGRGQARGRQDRGDIRHIQAEVQLHSGQGEKAGMGQLREQLASAFQEQMDVRRRLLELENRAMEVQIDTSRHLLTIAGWKHEKSRRALKWREEQRKECYAKDDSEKDSDTGDDQPDILEPPEVAAARESIAALVDEQKQLRKQKLALEQRCRELRARGRRLEETLPRRIGSEEQREVLSLLCRVHELEVENTEMQSHALLRDGALRHRHEAVRRLEQHRSLCDEIIQGQRQIIDDYNLAVPQRLEELYEVYLRELEEGSLEQATIMDQVASRALQDSSLPKITPAGTSLTPDSDLESVKTLSSDAQHLQNSALPPLSTESEGHHVFKAGTGAWQAKSSSVPTPPPIQLGSLVTQEAPAQDSLGSWINSSPDSSENLSEIPLSHKERKEILTGTKCIWVKAARRRSRALGTEGRHLLAPATERSSLSLHSLSEGDDARPPGPLACKRPPSPTLQHAASEDNLSSSTGEAPSRAVGHHGDGPRPWLRGQKKSLGKKREESLEAKRRKRRSRSFEVTGQGLSHPKTHLLGPHQAERISDHRMPVCRHPAPGIRHLGKVTLPLAKVKLPPSQNTGPGDSSPLAVPPNPGGGSRRATRGPRLPHGTSTHGKDGCSRHN

In an embodiment, CEP192 comprises the following sequence (SEQ ID NO:3):

1 agtgccctgg gacacctctt cagtccgtgg actttcccgc tgcacactgc cctccgaagt 61cggggacgcg ggctcgtgag atggaagatt ttcgaggtat agcagaagaa tcatttccaa 121gctttctcac caattcatta tttggtaaca gtgggatttt ggaaaatgtc actcifictt 181caaatcttgg cttgcctgtt gctgtttcta cacttgctag ggatagatcc agcactgata 241acaggtatcc tgatatccag gcatcttact tagtagaagg gagattttca gttccatccg 301ggtcatctcc cggaagccag agtgatgctg aaccaagaga gaggttacag cttagcttcc 361aggatgatga ttctatctct aggaaaaaga gctatgtgga aagtcaacgt ttgtcaaatg 421ctctcagcaa acagtcagct ttacaaatgg agacagcagg accagaagag gagccagccg 481gagctacaga atccttgcag ggccaagatc tcttcaacag ggcttcacca ctggaacaag 541cacaagactc acctattgat ificatttac agtcatggat gaataataag gaacccaaga 601ttgttgtgct tgatgctgga aaacattttg aagacaagac tctaaagagt gacctaagcc 661acactagctt attagaaaat gagaaactta tcttaccgac aagettggaa gattcttctg 721atgatgatat tgatgatgaa atgttttatg atgatcattt ggaggcttat tttgaacaac 781tggcaattcc aggaatgata tatgaagacc tagaaggacc agaacctcca gaaaaaggtt 841ttaagttacc tacaaatggt cttagacagg caaatgaaaa cggtagctta aactgcaagt 901ttcaatcaga aaataacagc tctctgattt ccctcgactc acactcttct gaaacaactc 961acaaagagtc tgaggaaagc caagttattt gtctacctgg gactagtaat tctataggta 1021ctggagatag tagaaggtac acagatggta tgttaccatt ttcctctggt acttggggaa 1081ctgagaaaga aatagaaaat ttgaagggta ttgttccaga tcttaacagt gaatgtgcaa 1141gtaaagatgt tctggtgaag accctcaggg ctattgatgt gaaacttaac tctgataatt 1201ttcatgatgc aaatgccaat agaggtggtt ttgatctgac tgaccctgta aaacaggggg 1261cagagtgtcc tcaccaaaat aagacagttt tgcacatgga tggatgttta gacactgaga 1321ctcctacggt gtccattcaa gaaaatgtgg atgtagcctc tttgaagccc attagtgaca 1381gtggaattaa tttcactgat gccatttggt caccaacttg tgaaaggcga acatgtgaat 1441gtcacgagtc catcgaaaag aataaagaca aaacagatct cccacagagt gtggtctatc 1501aaaatgaaga gggtaggtgg gtcacagacc ttgcctatta cacatctttt aatagcaaac 1561aaaatttaaa tgtgtctcta agtgatgaga tgaatgaaga cttcagatct ggttctgaag 1621catttgattt gattgcacaa gatgaagaag aatttaataa agagcatcaa tttatacagg 1681aagaaaacat agatgctcat aatacttcgg ttgcactggg cgatacgtcc tggggagcta 1741caattaatta cagtctgttg aggaaatcac gtagcacatc agatttggat aaagatgatg 1801ccagttattt acgtctgtct ttaggagagt tctttgctca aagatctgaa gctcttggtt 1861gccttggtgg tggtaacaat gtgaaaagac catcatttgg ctattttatt agatcaccag 1921agaagagaga acctattgcc ttaataagaa aatctgatgt atcaagaggt aatttggaaa 1981aagaaatggc tcatcttaac catgatctat attcaggaga tttaaatgaa cagtcccagg 2041cacagctaag tgaaggatca attacacttc aggttgaagc agtagagagt acttcacaag 2101tggatgaaaa tgatgtgacg ttaacggctg ataaaggcaa aacagaggac actttcttca 2161tgagcaacaa accccaaaga tacaaagaca agctaccaga tagtggtgat tctatgctta 2221ggatcagcac cattgcttca gccattgcag aggcatcagt taatactgat ccttcccaac 2281ttgctgcaat gatcaaggca ctttcaaata aaaccagaga caagactttt caggaagatg 2341agaaacaaaa ggactattct catgtgcgtc atttcttacc taatgattta gaaaaaagta 2401atggatccaa tgcacttgat atggagaaat accttaaaaa aacagaagtt agtagatatg 2461aaagtgcatt ggaaaacttt tcaagggcta gtatgtctga tacttgggat ttatctttgc 2521ccaaagaaca aactactcaa gacattcatc cggtggactt aagtgctact agtgtaagtg 2581tgagggcacc agaagaaaac acagcagcta ttgtttatgt tgaaaatgga gagagtgaga 2641atcaagagtc atttagaacc ataaactcct caaattcagt tacaaataga gagaataaca 2701gtgcagtagt tgatgtgaag acatgttcca ttgacaacaa attacaagat gttggtaacg 2761atgaaaaagc tacctcaatt tccactccat ctgatagtta ttcatcagtg aggaacccca 2821gaataacatc cctttgtctg ttaaaagact gtgaagaaat acgagataac agagaaaatc 2881agaggcaaaa tgagtgtgtc agtgaaataa gcaacagtga gaagcatgtg acttttgaaa 2941accatcgcat agtctcacct aaaaatagtg atttgaaaaa tacctctcct gagcatggtg 3001gacgtggctc agaggatgag caggagagct tcagaccttc cacgtcacca ctgagtcatt 3061cttctcctag tgaaatttct ggaacgagtt catcagggtg tgcgttagag tccifiggtt 3121cagcagctca gcagcagcag cctccctgtg agcaggagtt gtctcccttg gtgtgctcgc 3181ctgctggggt gagcaggctg acgtatgtgt ctgaaccaga gagctcctat cctaccacag 3241ccacagatga tgccctggag gaccgcaaga gtgatatcac cagcgagttg agtaccacaa 3301ttattcaagg cagtccagcc gcattggagg aacgggctat ggaaaaattg agagaaaaag 3361ttccatttca gaatagagga aaaggaacat tatcatctat tatccagaat aactctgata 3421caagaaaagc aactgaaact acttctctga gtagcaagcc tgaatatgta aaacctgact 3481ttagatggag taaagatcct tcctccaaaa gtggaaatct gttggaaacc agtgaggtag 3541gttggacatc aaaccctgag gaattggacc cgatcaggct ggctctcctg ggcaagtcag 3601gtctgagctg tcaggtgggg tcagccacat cacaccctgt gtcctgccag gagcctatag 3661atgaagatca aagaataagt cctaaagata agtcaactgc tggccgtgag ttcagtggcc 3721aggifictca tcagaccacc tctgaaaacc agtgtactcc tattcccagc agcacagttc 3781acagctctgt ggctgacatg cagaacatgc ctgctgctgt gcacgcactc ttgacacaac 3841cctctctcag cgctgctcct tttgctcagc ggtatttggg aacactccct tcaactggaa 3901gcaccacctt gcctcagtgc catgctggca atgccacagt ctgtggcttc tcaggaggcc 3961ttccctatcc agctgttgca ggagagcctg tgcagaactc tgtggctgtg ggaatttgtc 4021taggatcaaa tatcggctct ggatggatgg gtacctcttc cctctgtaac ccatattcta 4081ataccttaaa tcagaacctg ctaagcacaa caaaaccttt tcctgtgccg tctgttggta 4141caaactgtgg aattgaacca tgggattcag gagtgacatc aggattgggg agtgtccgag 4201tgcccgagga gttgaagctt cctcatgctt gctgtgtcgg gatcgcttcc cagaccctcc 4261tcagtgtgct taatccaact gaccgctggc tgcaagtcag cattggggtc ctcagcatta 4321gtgttaatgg tgaaaaggtg gatctttcaa catatcgttg tttagttttc aagaataaag 4381ccatcataag acctcatgcc acagaagaga taaaagtgct ttttatacca tccagtcctg 4441gggttttcag atgcacattc agtgttgctt cttggccatg ttcgacagat gctgagacca 4501tcgtacaggc agaagctttg gccagcaccg tcactctcac tgccattgcc gagagtcctg 4561ttattgaggt agaaacagaa aagaaagacg ttcttgattt tggtgacttg acttatggag 4621gctggaaagc cctcccacta aaattgataa accgaacgca tgccactgtg ccaattagac 4681tgattattaa tgctaacgct gtagcctggc gctgtttcac gttttccaag gaatccgtcc 4741gagctcctgt ggaagttgct ccttgcgctg atgtggtcac tcggctagca ggcccttctg 4801tggtcaacca catgatgcct gctagttatg atggacagga tccagaattt ctgatgattt 4861gggttctttt ccatagtcca aagaaacaga tcagctcttc agatattctg gactcagcag 4921aagaattctc ggcaaaagtt gatatcgaag ttgacagccc aaaccctacg cccgttctta 4981gaagtgtgag tctccgagca agagcaggaa tagctaggat ccatgctccc agggacttgc 5041agacgatgca tttcttggcc aaagtggctt cctcaagaaa gcagcactta cctttgaaaa 5101atgctgggaa cattgaagtt tatttggata tcaaggtccc agaacaagga agtcactttt 5161cagtggatcc aaagaatcta ctccttaaac ctggagaaga acatgaggtt attgtttcat 5221ttactccaaa ggatcctgaa gcctgcgagg aaaggatctt gaaaatattt gtgcagccat 5281ttggacctca gtatgaggta gtgttaaaag gcgaagtcat ttcttcagga agtaaacctc 5341tgtcacctgg accttgctta gatattccat cgattttgtc caacaaacaa tttctggctt 5401ggggaggagt ccctctaggt agaacacagc ttcagaaact agctttaaga aataattctg 5461catctacaac tcaacattta cgactgctta ttagaggaca agatcaggac tgctttcagc 5521ttcagaacac ttttggttca gaacagcgat tgaccagtaa ctgtgagatc agaattcacc 5581caaaggaaga cattttcatc tctgtattat ttgcacctac tcgattatct tgcatgttgg 5641ctagactaga aatcaaacaa cttggaaatc gatcacaacc aggcattaag ttcacaatac 5701ctttgtctgg atatggagga acaagcaatc ttattttgga aggcgttaaa aaattatctg 5761acagttacat ggtaacagtg aatggcttag tacctggcaa agaaagtaaa attgtttttt 5821ctgtccgcaa cactggctcc cgagcagctt ttgttaaagc agtaggtttt aaggattctc 5881agaaaaaagt tttgctggat cctaaagtat tgaggatttt tccagataaa tttgtactca 5941aggaaagaac acaagaaaat gttactttaa tatataatcc atcagacaga ggaatcaata 6001ataaaactgc aacagaacta tcaactgtat acttatttgg tggagatgaa atttcaagac 6061agcagtatcg cagggccctg ttacataaac cagagatgat aaaacagata cttccagaac 6121atagtgtgct tcaaaacatt aattttgttg aagcatttca agatgagcta ttagtaactg 6181aagtatatga tcttccccaa cgacctaatg atgttcagct cttttatgga agcatgtgta 6241aaattatact ttcagtaatt ggagaattca gagattgcat ttctagcaga gaattccttc 6301agccttcttc caaagctagc ttggaatcta caagcgactt gggagcttct gggaaacatg 6361gtggcaacgt ctctttggat gttttaccag tcaaaggtcc tcagggttct cctcttctct 6421cacgggcggc tcgcccgcct ctggatcagc tggcctccga agagccgtgg actgtcctac 6481ccgagcactt gattctggta gctccttctc cttgtgacat ggcaaaaact ggacgtttcc 6541agattgtgaa taactctgtg aggttactga gatttgagct gtgctggcca gcgcattgcc 6601tcacagtcac gccgcagcat ggatgtgtcg cgccagagag taaactacaa attcttgtga 6661gtcctaattc ctccttatcc acaaaacagt caatgttccc gtggagtggt ttgatctata 6721tacactgtga cgatggacag aagaaaattg tgaaagttca aattcgagaa gatttaactc 6781aagtggaact tttaactcgt ttgacctcca aaccatttgg aattctttcc ccagtatctg 6841agccttcagt tagtcatttg gtcaaaccaa tgacaaaacc gccttccaca aaagttgaaa 6901taagaaacaa gagtattact tttcctacaa cagaacctgg tgaaacttca gagagctgtc 6961tagaactcga gaatcatggc accacagacg tgaaatggca tctgtcatct ttagcgccac 7021cttatgtcaa gggagttgat gaaagtggag atgffittag agctacctat gcagcattca 7081gatgttctcc tatttctggt ctgctggaaa gccatgggat ccaaaaagtc tccatcacat 7141tifigcccag aggtaggggg gattatgccc agttttggga tgttgaatgt caccctctta 7201aggagcctca catgaaacac acgttgagat tccaactctc tggacaaagc atcgaagcag 7261aaaatgagcc tgaaaacgca tgcctttcca cggattccct cattaaaata gatcatttag 7321ttaagccccg aagacaagct gtgtcagagg cttctgctcg catacctgag cagcttgatg 7381tgactgctcg tggagtttat gccccagagg atgtgtacag gttccggccg actagtgtgg 7441gggaatcacg gacacttaaa gtcaatctgc gaaataattc ttttattaca cactcactga 7501agtttttgag tcccagagag ccattctatg tcaaacattc caagtactct ttgagagccc 7561agcattacat caacatgccc gtgcagttca aaccgaagtc cgcaggcaaa tttgaagctt 7621tgcttgtcat tcaaacagat gaaggcaaga gtattgctat tcgactaatt ggtgaagctc 7681ttggaaaaaa ttaactagaa tacattffig tgtaaagtaa attacataag ttgtattttg 7741ttaactttat ctttctacac tacaattatg cttttgtata tatatifigt atgatggata 7801tctataattg tagattttgt ttttacaagc taatactgaa gactcgactg aaatattatg 7861tatctagccc atagtattgt acttaacttt tacaggtgag aagagagttc tgtgtttgca 7921ttgattatga tattctgaat aaatatggaa tatattttaa tgtggtatat ccagaaaaaa 7981aaaaaaaaaa aaaaa

In an embodiment, Cep192 gene product comprises the following sequence(SEQ ID NO:4):

MEDFRGIAEESFPSFLTNSLFGNSGILENVTLSSNLGLPVAVSTLARDRSSTDNRYPDIQASYLVEGRFSVPSGSSPGSQSDAEPRERLQLSFQDDDSISRKKSYVESQRLSNALSKQSALQMETAGPEEEPAGATESLQGQDLFNRASPLEQAQDSPIDFHLQSWMNNKEPKIVVLDAGKHFEDKTLKSDLSHTSLLENEKLILPTSLEDSSDDDIDDEMFYDDHLEAYFEQLAIPGMIYEDLEGPEPPEKGFKLPTNGLRQANENGSLNCKFQSENNSSLISLDSHSSETTHKESEESQVICLPGTSNSIGTGDSRRYTDGMLPFSSGTWGTEKEIENLKGIVPDLNSECASKDVLVKTLRAIDVKLNSDNFHDANANRGGFDLTDPVKQGAECPHQNKTVLHMDGCLDTETPTVSIQENVDVASLKPISDSGINFTDAIWSPTCERRTCECHESIEKNKDKTDLPQSVVYQNEEGRWVTDLAYYTSFNSKQNLNVSLSDEMNEDFRSGSEAFDLIAQDEEEFNKEHQFIQEENIDAHNTSVALGDTSWGATINYSLLRKSRSTSDLDKDDASYLRLSLGEFFAQRSEALGCLGGGNNVKRPSFGYFIRSPEKREPIALIRKSDVSRGNLEKEMAHLNHDLYSGDLNEQSQAQLSEGSITLQVEAVESTSQVDENDVTLTADKGKTEDTFFMSNKPQRYKDKLPDSGDSMLRISTIASAIAEASVNTDPSQLAAMIKALSNKTRDKTFQEDEKQKDYSHVRHFLPNDLEKSNGSNALDMEKYLKKTEVSRYESALENFSRASMSDTWDLSLPKEQTTQDIHPVDLSATSVSVRAPEENTAAIVYVENGESENQESFRTINSSNSVTNRENNSAVVDVKTCSIDNKLQDVGNDEKATSISTPSDSYSSVRNPRITSLCLLKDCEEIRDNRENQRQNECVSEISNSEKHVTFENHRIVSPKNSDLKNTSPEHGGRGSEDEQESFRPSTSPLSHSSPSEISGTSSSGCALESFGSAAQQQQPPCEQELSPLVCSPAGVSRLTYVSEPESSYPTTATDDALEDRKSDITSELSTTIIQGSPAALEERAMEKLREKVPFQNRGKGTLSSIIQNNSDTRKATETTSLSSKPEYVKPDFRWSKDPSSKSGNLLETSEVGWTSNPEELDPIRLALLGKSGLSCQVGSATSHPVSCQEPIDEDQRISPKDKSTAGREFSGQVSHQTTSENQCTPIPSSTVHSSVADMQNMPAAVHALLTQPSLSAAPFAQRYLGTLPSTGSTTLPQCHAGNATVCGFSGGLPYPAVAGEPVQNSVAVGICLGSNIGSGWMGTSSLCNPYSNTLNQNLLSTTKPFPVPSVGTNCGIEPWDSGVTSGLGSVRVPEELKLPHACCVGIASQTLLSVLNPTDRWLQVSIGVLSISVNGEKVDLSTYRCLVFKNKAIIRPHATEETKVLFIPSSPGVFRCTFSVASWPCSTDAETIVQAEALASTVTLTAIAESPVIEVETEKKDVLDFGDLTYGGWKALPLKLINRTHATVPIRLIINANAVAWRCFTFSKESVRAPVEVAPCADVVTRLAGPSVVNHMMPASYDGQDPEFLMIWVLFHSPKKQISSSDILDSAEEFSAKVDIEVDSPNPTPVLRSVSLRARAGIARTHAPRDLQTMHFLAKVASSRKQHLPLKNAGNIEVYLDIKVPEQGSHFSVDPKNLLLKPGEEHEVIVSFTPKDPEACEERILKIFVQPFGPQYEVVLKGEVISSGSKPLSPGPCLDIPSILSNKQFLAWGGVPLGRTQLQKLALRNNSASTTQHLRLLIRGQDQDCFQLQNTFGSEQRLTSNCEIRTHPKEDIFISVLFAPTRLSCMLARLEIKQLGNRSQPGIKFTIPLSGYGGTSNLILEGVKKLSDSYMVTVNGLVPGKESKIVFSVRNTGSRAAFVKAVGFKDSQKKVLLDPKVLRIFPDKFVLKERTQENVTLIYNPSDRGINNKTATELSTVYLFGGDETSRQQYRRALLHKPEMIKQILPEHSVLQNINFVEAFQDELLVTEVYDLPQRPNDVQLFYGSMCKIILSVIGEFRDCISSREFLQPSSKASLESTSDLGASGKHGGNVSLDVLPVKGPQGSPLLSRAARPPLDQLASEEPWTVLPEHLILVAPSPCDMAKTGRFQIVNNSVRLLRFELCWPAHCLTVTPQHGCVAPESKLQILVSPNSSLSTKQSMFPWSGLIYIHCDDGQKKIVKVQIREDLTQVELLTRLTSKPFGILSPVSEPSVSHLVKPMTKPPSTKVEIRNKSITFPTTEPGETSESCLELENHGTTDVKWHLSSLAPPYVKGVDESGDVFRATYAAFRCSPISGLLESHGTQKVSITFLPRGRGDYAQFWDVECHPLKEPHMKHTLRFQLSGQSIEAENEPENACLSTDSLIKIDHLVKPRRQAVSEASARIPEQLDVTARGVYAPEDVYRFRPTSVGESRTLKVNLRNNSFITHSLKFLSPREPFYVKHSKYSLRAQHYINMPVQFKPKSAGKFEALLVTQTDEGKSIAIRLIGEALGKN

The phrase “and/or” as used herein, with option A and/or option B forexample, encompasses the individual embodiments of (i) option A alone,(ii) option B alone, and (iii) option A plus option B.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group subjectly and all possible subgroups of the main group, butalso the main group absent one or more of the group members. The presentinvention also envisages the explicit exclusion of one or more of any ofthe group members in the claimed invention.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

In the event that one or more of the literature and similar materialsincorporated by reference herein differs from or contradicts thisapplication, including but not limited to defined terms, term usage,described techniques, or the like, this application controls.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Introduction

Cell motility is driven by a cycle of protrusion of the membrane at thecell front, adhesion of the protrusion to the substratum, contractilityto move the cell body forward, and finally disadhesion at the rear.While the roles of the actin cytoskeleton in these events have beenstudied in detail (Ridley, Schwartz et al. 2003; Gardel, Schneider etal. 2010), much less is known about the specific contributions ofmicrotubules. It is, however, clear that the microtubule cytoskeleton isrequired for the normal polarization and motility of many cell types andthere is emerging evidence that it does so by exerting spatiotemporalcontrol over actin dynamics/contractility and the delivery of membraneand signaling molecules to the cell periphery (Rodriguez, Schaefer etal. 2003; Small and Kaverina 2003; Watanabe, Noritake et al. 2005).Microtubules also contribute to cell migration by regulating thedisassembly of focal adhesions (Broussard, Webb et al. 2008) (FAs). FAsare integrin-based macromolecular assemblies that link the actincytoskeleton to extracellular matrix and thus anchor the cell to itssubstratum to provide traction for cell motility. The primarymicrotubule nucleating and organizing structure in the cell is thecentrosome. Herein it is disclosed that Cep192 and Kif19 regulate themicrotubule cytoskeleton.

While it would be difficult to raise the levels of a particularregulatory protein rapidly in relevant cells, it is far more tractableto lower the levels of a target protein through RNA interference (RNAi).The essence of this approach is to inhibit messenger RNA (mRNA) fromcoding for the synthesis of a target protein. There are various types ofRNAi, such as plasmid-driven shRNA, which has the advantage of beingtargetable to certain cell populations and is generally long-lasting interms of suppression of protein expression. shRNA generally requires aspecialized transfection technique such as viral entry orelectroporation. Small interfering RNAs (siRNAs) do not involve the useof a plasmid, are tiny and hence can more readily be introduced intocells, and offer more flexibility in terms of target sequences. siRNAcan be handled and treated much like a drug and theoretically caninterfere with the translation of almost any mRNA as long as the mRNAhas a distinctive sequence. Therefore, siRNA has far broader flexibilitythan traditional drugs. A key to capitalizing on the therapeuticbenefits of siRNA lies in effective delivery systems. Carriers such asnanoparticles have now become the approach of choice. Nanotechnology isbroadly considered the study of manipulations of materials at thenanometer scale, roughly 1 to 500 nm. Materials at this scale possess ahigher surface to volume ratio and, as a result, their physicalproperties tend to be different from materials at the macro or microscale. Novel properties that result from such modifications have led toapplications in fields such as catalysis, microelectronics, robotics andmedicine. The medical and biological applications are particularlyinteresting because most biochemical processes, especially thoseinvolving macromolecules, occur at the lower end of the nano scale.Nanotechnology, therefore, holds the promise of being able to duplicatebiochemical processes and directly alter these processes using man-madematerials. With the progress of material synthesis and the rise ofnanotechnology, the generation of nanomaterials with specific functionshas become possible. In to, for example, solid tumors in humans.

To date, the reported liposomal and other nanoparticle based deliveryvehicles for siRNA have involved systemic delivery. In contrast, a noveldelivery approach disclosed herein is effective for both topical andsystemic applications. One preferred embodiment of the platform is basedon a hydrogel/sugar glass composite, or hybrid nanoparticle platformcapable of encapsulating and controllably releasing a broad range oftherapeutically relevant materials ranging from gaseous nitric oxide topeptides to larger macromolecules such as chemotherapeutic agents andphosphodiesterase inhibitors. The versatility of this biocompatible andnontoxic platform has been shown in pre-clinical studies demonstrating:i) topical efficacy in clearing both Gram positive and negativecutaneous wound infections, accelerating wound healing, and promotingerectile activity; and ii) systemic efficacy in modulatingcardiovascular parameters.

The data herein indicates that kif19 and Cep192 proteins exert profoundregulatory control over the motility and/or growth characteristics ofkey cells required for wound closure, re-vascularization andre-innervation. Each protein can be targeted independently by differentnp-si to control a distinct aspect of the wound healing cascade. Thenanoparticle platform can be very effective as a topical deliveryvehicle for the siRNA. The preferred therapeutic platform technology isnanoparticle-encapsulated siRNAs (np-si) targeting the expression ofCEP192 and KIF19 genes encoding regulators of the microtubulecytoskeleton. Without being bound be theory, it is understood that Kif19np-si treatments inhibit fibroblast motility to reducefibrosis/scarring. The Cep192 np-si treatment does similar, but alsoinhibits axonal growth to ameliorate the pain that results frompremature axon sprouting into wounded tissue.

The nanoparticle delivery system bypasses pitfalls typically associatedwith therapeutic siRNA—for example, the ability to deliver therapeuticlevels of siRNA to enhance the closure of surface wounds in vivo.

Example 1

Kif19 normally promotes cell motility by stimulating the disassembly ofintegrin-based adhesion complexes that link cells to the underlyingextracellular matrix. siRNA-mediated depletion of Kif19 inhibits 1)cancer cell motility in vitro; 2) matrigel invasion of primary tumorcells ex vivo, 3) and movement of cells into excision wounds in mice.

FIG. 1 shows a confocal micrograph showing a human U2OS cell doublelabeled for Kif19 and the FA protein, vinculin. The far right panel is ahigher magnification of the region boxed in “merge”. As shown in FIGS. 2and 3, the depletion of Kif19 from tissue culture cells induces anincrease in focal adhesion size and stability. FIG. 2 shows regions ofU2OS cells (human Osteosarcoma) immunostained for the focal adhesionprotein vinculin. The depletion of Kif19 by siRNA induces a substantialincrease in the size and number of focal adhesions particularly in thecell interior. A time series was obtained showing theassembly/disassembly dynamics of focal adhesions in GFP-vinculinexpressing control and Kif19 siRNA-treated U2OS cells. In FIG. 3, panelA shows fluorescence recovery after photobleaching (FRAP) ofGFP-vinculin labeled focal adhesions from control and Kif19siRNA-treated U2OS cells. Panel B shows a representative fluorescencerecovery plot from each condition. Panel C plots the density of focaladhesions in untreated cells (pre) and at various time points afternocodazole washout. Repolymerization of MTs after nocodazole washout waspreviously shown to stimulate focal adhesion disassembly (Ezratty,Partridge et al. 2005). The depletion of Kif19 prevents the disassemblyof focal adhesions after nocodazole washout. The siRNA sequences usedfor Kif19 are as follows:

(SEQ ID NO: 5) 5′-GGAAGUAAGCUCAGGAUCUCAGCAG-3′ (SEQ ID NO: 6)5′-GUCCUUCAUUCGAGUCCUAUAGUCGUC-3′.

FIG. 4 shows siRNA depletion of Kif19 decreases the motility of cancercells in vitro. Depletion of Kif19 prevents tumor cell invasion fromanaplastic thyroid carcinomas embedded in matrigel. FIG. 5 shows ananaplastic thyroid carcinoma mouse model (Archiuch, Rousseau et al,Oncotarget, December 2011). Accounts for 40% of all thyroid cancerdeaths and extremely metastatic. Nearly 100% lethality, median survival4 months. Currently not treatable. Dissociated tumor is removed frommouse and bathed in nanoparticles containing control or Kif19 siRNA for2-24 hrs. Tumors are then embedded in matrigel and imaged daily.Movement of cells from tumor into matrigel is considered invasion. Blackdots moving away from the dark central mass are invasive tumor cells.Kif19 nanoparticle siRNA treatment reduces tumor cell invasion relativeto controls.

Depletion of Kif19 was later confirmed to inhibit cell movement intomouse full thickness biopsy wounds as compared to control.

Kif-19 depolymerizes microtubules in vitro, as shown in FIG. 6 where atime-series of T1RF images shows a field of fluorescently-labeledtaxol-stabilized microtubules incubated with purified recombinantfull-length Kif19. The time from the first to last image is 5 minutes.

In summary, Kif19 is a microtubule depolymerizing enzyme in vitro thatlocalizes to and stimulates the turnover of substrate adhesions in cells(FIG. 7A-D). siRNA depletion of Kif19 in human epithelial and fibroblastcell models nearly completely suppresses cell motility likely becausethese cells become too tightly attached to their underlying substratum.Kif19 is the first and, at present, only microtubule regulatory proteinknown to be housed within the substrate adhesion complex. Agents thatsuppress Kif19, such as Kif19 siRNA nanoparticles, can be used as ameans to prevent fibrosis/scarring later in the wound healing process.

Example 2

Cep192 promotes cell motility via the nucleation of centrosomalmicrotubules. Cep192 is a centrosomal scaffolding protein required forthe nucleation of microtubules from centrosomes. siRNA-mediateddepletion of Cep192 inhibits 1) the motility of cancer cells and primaryhuman keratinocytes in vitro; 2) matrigel invasion of primary tumorcells ex vivo; 3) axon outgrowth from primary neurons. This additionallyidentifies Cep192, over Kif19, as a therapeutic target for mitigation ofpain after wounding.

The centrosome is an organelle that serves as the main microtubuleorganizing center (MTOC) of the animal cell as well as a regulator ofcell-cycle progression. Centrosomes are composed of two orthogonallyarranged centrioles surrounded by an amorphous mass of protein termedthe pericentriolar material (PCM). The PCM contains proteins responsiblefor microtubule nucleation and anchoring.

Cep192 is a centrosome scaffolding protein required for centrosomalmicrotubule nucleation during mitosis (Gomez-Ferreria, Rath et al. 2007;Gomez-Ferreria and Sharp 2008). Disclosed herein is that Cep192 is alsorequired for the nucleation of centrosomal microtubules in interphasecells. Depletion of Cep192 strongly suppresses the motility of bothcancer and skin cells and thus Cep192 is a novel target foranti-metastatic and anti-fibrotic therapeutics. Additionally, depletionof Cep192 inhibits axon outgrowth from primary adult rat dorsal rootganglion neurons. Thus, Cep192 can also be targeted to suppressexcessive early axon sprouting known to be associated with pain.

Cep192 is found to localize to centrosomes in interphase cells and isrequired for normal microtubule organization (see FIG. 8). Cep192 wasalso found to stimulate microtubule nucleation from centrosomes (FIG.9).

It was found that Cep192 is required for normal cell motility in vitro.FIG. 10 shows in 10A) time-lapse phase-contrast images of control andCep192 siRNA treated U2OS cells from an in vitro wound healing assay.U2OS cells were plated into Ibidi Culture-Insert dishes followingknockdown. In 10B), significantly fewer Cep192 depleted cells enteredthe wound zone relative to controls. P<0.0001. S.E.M. is depicted asvertical bars. FIG. 10C) shows time-lapse phase-contrast images ofcontrol and Cep192 siRNA treated HEKa (human epidermalkeratinoctyes—adult) cells from an in vitro a wound healing assay. HEKacells were plated into Ibidi Culture-Insert dishes following. 10D) Showssignificantly fewer Cep192 depleted cells entered the wound zonerelative to controls. P<0.0001. S.E.M. is depicted as vertical bars. ThesiRNA sequences used for Sep192 are as follows:

(SEQ ID NO: 7) 5′-CACAUGAUGCCUGCUAGUU-3′ (SEQ ID NO: 8)5′-GACACUUUCUUCAUGAGCA-3′ (SEQ ID NO: 9) 5′-GGACUUAAGUGCUACUAGU-3′.

Depletion of Cep192 prevents tumor cell invasion and metastasis. Theeffects on anaplastic thyroid carcinoma are shown in FIG. 11 and theeffects on large cell lung tumor are shown in FIG. 12.

Depletion of Cep192 inhibits axon outgrowth from primary rat neurons.FIG. 13 shows fidgetin and Cep192 regulate axon regeneration. Images areimmunofluorescence micrographs of primary adult rat DRG neurons treatedwith control, Fidgetin or Cep192 nanoparticle encapsulated siRNA. Cellswere fixed 24 hours after plating and siRNA treatment. Bottom rightpanel shows the average axon length in each condition (longest processfrom each individual cell was measured; error bars are SEM). ***P<0.01;**P<0.05. In contrast to Fidgetin np-si, Cep192 np-si treatmentssuppress axon regrowth in adult DRG neurons—Cep192 and Fidgetin arelikely functionally antagonistic in this regard. Agents that suppressCep192, such as Cep192 np-si, can be used to suppress excessive earlyaxon sprouting known to be associated with pain.

Example 3

Angiogenesis of fetal hearts 48 hours after treatment: As shown in FIG.14, Cep192 siRNA-treated hearts two days after siRNA treatment have noapparent vessels. In the control, however, migrating endocaridal cellshave penetrated the ventricular wall and formed a fine vascular network.Thus, the depletion of Cep192 dramatically inhibits the angiogenicprocess by the endocardial cells.

Methods

Np-si application for experiments: This can be performed by mixing thenanoparticles in either sterile saline or water to achieve the targetedconcentration in no more than 10 ul aliquots. The solution is applieddirectly to the wound, or target area, where it is rapidly absorbed.Controls include i) non-specific siRNA nanoparticles and ii) water orsaline alone. Two different treatment regimens are exemplified here: Inregimen 1, np-si are administered daily beginning 30 minutes afterwounding though day 8. In regimen 2, np-si are administered every otherday beginning 30 minutes after wounding (day 0, 2, 4, 6, and 8).

Np-si formulation. For the nanoparticles a hydrogel-based nanoparticleplatform is used. A final concentration of siRNA of 0.30 to 0.35 nmoleper mg of dry nanoparticles is used in studies. siRNAs are anionic butcationic stabilization is preferred for nanoparticle encapsulation andsiRNA stability. In an embodiment, the formulation utilizes the cationicpolysaccharide chitosan as a stabilizing factor for the siRNA. Thecationic character of the nanoparticles can be enhanced by doping theformulation with varying amounts of positively charged amino silanes.

PEGylation of the np-si: Increasing the size of PEG moleculesincorporated into the formulation may increase the rate of release forsiRNA (this is determined using fluorescent labeled siRNA).Post-preparative PEGylaton of the np-si can be means of furtherminimizing aggregation and improving in vivo lifetime. In an embodiment,the conjugation of functionalized PEG chains (PEG-500/PEG-3000/PEG-5000)to the surface of np-si in alcohol/water medium to minimize the leakageof siRNA from the particles is effected.

Wound healing determination: Photographs of the wounds are taken dailyto follow gross visual wound healing as assessed by the area of thewound uncovered by the migrating epithelia. Each wound is measured dailyusing a caliper and the area is determined.

Morphometric analysis of wound sections: Wound re-epithelialization ismeasured in Hematoxylin and Eosin stained sections from the center ofthe wound. The distance between the wound edges, defined by the distancebetween the first hair follicle encountered at each end of the wound,and the distance that the epithelium had traversed into the wound, isanalyzed using ImageJ. The percentage of re-epithelialization [(distancetraversed by epithelium)/(distance between wound edges)×100] iscalculated and averaged for two sections per wound.

Collagen deposition: Staining is performed using Masson's trichromestain and the percentage of blue collagen-stained area relative to thetotal area of the wound bed after taking digital images. This isquantified by counting the number of pixels staining above a thresholdintensity and normalizing to the total number of pixels.

Proliferation rate. To visualize cell proliferation, mice are injectedintrapertonially (120 mg/kg BrdU (Sigma-Aldrich, USA)) 2-4 hrs. prior tosacrifice and cutaneous wounds are harvested for paraffin embedding andBrdU immunohistochemistry. Tissue sections will be deparaffinized andrehydrated through graded alcohols and incubated overnight at roomtemperature with a biotinylated monoclonal BrdU antibody (Zymed, SouthFrancisco, Calif.).

Nuclear staining are visualized using Streptavidin-peroxidase anddiaminobenzidine (DAB) and samples will be lightly counterstained withhematoxylin. Wound tissue from mice that were not injected with BrdU isused as a negative control. Digital photographs are taken at high(40-60×) magnification (Zeiss AxioHOME microscope) and epithelial cellssections are examined using ImageJ software and classified as BrdUpositive if they grossly demonstrated brown-stained nuclei from DABstaining or as BrdU negative if they were blue stained. nuclei. Theproliferation rate is then calculated as the percentage of BrdU positivecells over the total number of cells within the ROI.

Angiogenesis: Wound sections are stained using CD31 antibody (alsocalled platelet-derived endothelial cell adhesion molecule-1). Digitalimages at 40× magnification covering the majority of the wound bed aretaken and the percent area stained in each image are quantified bycounting the number of pixels staining above a threshold intensity andnormalizing to the total number of pixels. Threshold intensity will beset such that only clearly stained pixels are counted. Stainingidentified as artifact, large vessels, and areas deemed to be outsidethe wound bed will be excluded.

Reinnervation: Wound sections post injury days 7 and 14 will be stainedfor protein gene product 9.5 (PGP9.5), a pan-neuronal marker, and thesensory neuropeptides calcitonin gene related peptide (CGRP) andsubstance P (SP). Nerve fiber growth into the wounds is compared betweencontrol and treated wounds,

Histopathology of epidermal stem cells and the stem cell niche at thehair bulge. For identification of epidermal stem cells in variouscohorts of animals, immunohistochemistry is performed for the followingmarkers of epidermal stem cells-CD34, Cytokeratin 15, BmiI, LrigI,BlimpI, Nestin, Lgr5, CD-200, β1-Integrin, according to publishedreports. The epidermal stem cell niche is characterized byimmunohistochemistry for α-smooth muscle actin (α-SMA) to detectepidermal myofibroblast and vascular smooth muscle cells, ICAM-1 forendothelial cells, F4/80 for macrophages.

Methods for angiogenesis experiments: Hearts were dissected fromembryonic day (E) 11.5 Nfatc1-Cre; eGFP embryos, bathed 2 hours withsiRNA and placed onto a 3D matrigel supplement with 10 ng/ml VEGF-A.Images (e.g. see FIG. 14) were then taken every day to record thecoronary angiogenesis by eGFP marked endocardial cells.

REFERENCES

-   Arciuch et al., “Thyrocyte-specific inactivation of p53 and Pten    results in anaplastic thyroid carcinomas faithfully recapitulating    human tumors,” Oncogene, Vol 2, No 12: 1109-1126 (2011).-   Broussard, J. A., D. J. Webb, et al. (2008). “Asymmetric focal    adhesion disassembly in motile cells.” Curr Opin Cell Biol 20(1):    85-90.-   Efimov, A. and I. Kaverina (2009). “Significance of microtubule    catastrophes at focal adhesion sites.” Cell Adh Migr 3(3): 285-287.-   Efimov, A., N. Schiefermeier, et al. (2008). “Paxillin-dependent    stimulation of microtubule catastrophes at focal adhesion sites.” J    Cell Sci 121(Pt 2): 196-204.-   Ezratty, E. J., M. A. Partridge, et al. (2005). “Microtubule-induced    focal adhesion disassembly is mediated by dynamin and focal adhesion    kinase.” Nat Cell Biol 7(6): 581-590.-   Gardel, M. L., I. C. Schneider, et al. (2010). “Mechanical    integration of actin and adhesion dynamics in cell migration.” Annu    Rev Cell Dev Biol 26: 315-333.-   Gomez-Ferreria, M. A., U. Rath, et al. (2007). “Human Cep192 is    required for mitotic centrosome and spindle assembly.” Current    biology: CB 17(22): 1960-1966.-   Gomez-Ferreria, M. A. and D. J. Sharp (2008). “Cep192 and the    generation of the mitotic spindle.” Cell cycle 7(11): 1507-1510.-   Ridley, A. J., M. A. Schwartz, et al. (2003). “Cell migration:    integrating signals from front to back.” Science 302(5651):    1704-1709.-   Rodriguez, O. C., A. W. Schaefer, et al. (2003). “Conserved    microtubule-actin interactions in cell movement and morphogenesis.”    Nat Cell Biol 5(7): 599-609.-   Small, J. V. and I. Kaverina (2003). “Microtubules meet substrate    adhesions to arrange cell polarity.” Curr Opin Cell Biol 15(1):    40-47.-   Watanabe, T., J. Noritake, et al. (2005). “Regulation of    microtubules in cell migration.” Trends Cell Biol 15(2): 76-83.

What is claimed is:
 1. A method of treating metastasis or inhibitingmetastasis in a subject having osteosarcoma comprising administering tothe subject an amount of an siRNA or shRNA inhibitor of KIF19 effectiveto treat metastasis or inhibit metastasis of osteosarcoma.
 2. The methodof claim 1, wherein the KIF19 is human KIF19.
 3. The method of claim 1,wherein the siRNA directed to KIF19 is administered.
 4. The method ofclaim 1, wherein the shRNA directed to KIF19 is administered.
 5. Themethod of claim 3, wherein the siRNA is administered as a compositioncomprising the siRNA encapsulated completely or partially by ananoparticle.
 6. The method of claim 4, wherein the shRNA isadministered as a composition comprising the shRNA encapsulatedcompletely or partially by a nanoparticle.
 7. The method of claim 5,wherein the nanoparticle is a liposomal nanoparticle.
 8. The method ofclaim 6, wherein the nanoparticle is a liposomal nanoparticle.
 9. Themethod of claim 5, wherein the nanoparticle is pegylated.
 10. The methodof claim 6, wherein the nanoparticle is pegylated.
 11. The method ofclaim 3, wherein the siRNA is administered as a viral vector.
 12. Themethod of claim 4, wherein the shRNA is administered as a viral vector.13. The method of claim 3, wherein the siRNA is modified at a 2 positionof a sugar of at least one nucleotide thereof of at least one strandthereof.
 14. The method of claim 13, wherein the siRNA is modified witha 2′-OMe.
 15. The method of claim 3, wherein the siRNA is administeredas a composition comprising the siRNA encapsulated in a nanoparticle.16. The method of claim 4, wherein the shRNA is administered as acomposition comprising the shRNA encapsulated in a nanoparticle.
 17. Themethod of claim 3 wherein the siRNA is selected from SEQ ID NO:5 and SEQID NO:6.
 18. The method of claim 4, wherein the shRNA is modified at a 2position of a sugar of at least nucleotide thereof of at least onestrand thereof.
 19. The method of claim 18, wherein the shRNA ismodified with a 2′-OMe.